WO2011017160A1 - Protéines et gènes d'interférons humains mutant - Google Patents

Protéines et gènes d'interférons humains mutant Download PDF

Info

Publication number
WO2011017160A1
WO2011017160A1 PCT/US2010/043536 US2010043536W WO2011017160A1 WO 2011017160 A1 WO2011017160 A1 WO 2011017160A1 US 2010043536 W US2010043536 W US 2010043536W WO 2011017160 A1 WO2011017160 A1 WO 2011017160A1
Authority
WO
WIPO (PCT)
Prior art keywords
interferon
virus
subject
sequence identity
nucleotides
Prior art date
Application number
PCT/US2010/043536
Other languages
English (en)
Inventor
William A. Clark
Original Assignee
Pestka Biomedical Laboratories, Inc.
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 Pestka Biomedical Laboratories, Inc. filed Critical Pestka Biomedical Laboratories, Inc.
Publication of WO2011017160A1 publication Critical patent/WO2011017160A1/fr

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]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to interferons, mutant interferons and biologically active fragments of interferons.
  • the invention also relates to methods of using interferons, mutant interferons and biologically active fragments of interferons in a pharmaceutical composition and for treating a subject infected with a virus.
  • Interferons are a well known family of cytokines secreted by a large
  • IFN- ⁇ known also as immune interferon, is the only Type II interferon whereas the
  • Type I human interferons consist of several classes: IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN-K and IFN- ⁇ . There is only one human IFN- ⁇ and one human IFN- ⁇ , but a family of multiple IFN- ⁇ species exists. IFN- ⁇ is only found in ungulates; there is no human IFN- ⁇ . The IFNs exhibit anti- viral, immunoregulatory, and antiproliferative activity. The clinical potential of interferons has been recognized.
  • IFN- ⁇ s homogeneous leukocyte interferons
  • CML chronic myelogenous leukemia
  • interferons are a family of proteins characterized by a potent ability to confer a virus-resistant state in their target cells.
  • interferon can inhibit cell proliferation, modulate immune responses and alter expression of proteins.
  • interferons were natural molecules produced by normal individuals. Indeed, the specific thesis was that all the interferons prepared for clinical use, be they natural- or recombinant-generated products, represented interferons that were produced naturally by normal people. This is true for a large number of interferons as well as specific growth factors, lymphokines, cytokines, hormones, clotting factors and other proteins that have been produced.
  • the invention comprises a biologically active interferon polypeptide or fragment thereof encoded by a mutant interferon polynucleotide, wherein the interferon polypeptide is selected from the group consisting of:
  • the biological activity may be selected from the group consisting of antiviral activity, antiproliferative activity, induction of interferon gamma in NK-92 cells, and MHC class I antigen expression induction activity.
  • the biologically active Interferon polypeptide or fragment thereof may also be used in a pharmaceutical composition with a suitable excipient.
  • the invention provides a method of treating a virus- infected subject or reducing a subject's risk of infection by a virus, comprising administering to the subject the biologically active interferon polypeptide or fragment thereof described above, wherein the virus is selected from the group consisting of severe acute respiratory syndrome-associated coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruscs, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, rotavirus, rabies, pap
  • SARS severe acute respiratory
  • the interferon polypeptide comprises any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.
  • the mutant interferon polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1 , 3, 5, 7, 9, 11, 13, 15, 17, and 19.
  • the subject may be a human being, non-human primate, feline, canine, fish, and farm animal.
  • the interferon may be administered nasally, orally, parenterally, topically, rectally, by injection, by inhalation, by eye lotion, by ointment, by suppository, by controlled release patch, by infusion, or by inhalation.
  • the interferon is administered to the subject's nasopharyngeal mucosa or lung epithelium.
  • the amount of the interferon polypeptide administered may be an amount effective to reduce the concentration of the virus particles in the subject.
  • the amount of the interferon polypeptide administered is an amount effective to prevent or reduce an increase in the concentration of virus particles in the subject.
  • Figure 1 Sequences of novel interferon genes (SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 17, and 19) and proteins (2, 4, 6, 8, 10, 12, 14, 16, 18, and 20).
  • Figure 2 Relative biological activities of novel interferon proteins in several bioassays in comparison to human IFN alpha 2a.
  • the effectiveness of IFN ⁇ 2a and novel interferons was tested in four separate bioassays. In all cases the activity was standardized to that of IFN ⁇ 2, which was set as one. If the IFN is more potent than IFN ⁇ 2, then the value is greater than one and if less potent, then the value is less than one.
  • Figure 3 Relative biological activities of each novel interferon protein in several bioassays in comparison to the congener wild-type interferon proteins. The effectiveness of IFN ⁇ 2a and novel interferons was tested in four separate bioassays. In all cases the activity was standardized to that of the congener wide type which was set as one. If the IFN is more potent than congener wide type, then the value is greater than one and if less potent then it is less than one.
  • nucleotide and amino acid sequences described herein within the sequence listing with respect to a nucleotide sequence, an "n” or “x” can refer to any nucleotide, whereas with respect to a protein sequence, an "n” or “x” can refer to any amino acid.
  • the term “comprising” is an open-ended term that includes the specific elements and may include additional, unrecited elements.
  • “Comprising” may be synonymous with “including” or “containing”.
  • “Comprising” may also, separately and independently of the above definition, be read as “consisting essentially of or “consisting of.
  • “consisting of” is a closed term that includes only the specific elements recited, and “consisting essentially of includes the specific elements recited and may include additional unrecited, nonmaterial elements.
  • sequence includes nucleotide and amino acid sequences.
  • a fragment is a portion of a polypeptide or polynucleotide sequence that is at least 2 amino acids in length or at least 6 nucleotides in length, respectively.
  • oligopeptide refers to an amino acid sequence between 2 and about 20 amino acids in length.
  • oligonucleotide refers to a nucleotide sequence between 2 and about 50 nucleotides in length.
  • farm animal includes, but is not limited to, artiodactyla, lagomorpha, carnivora, reptilia, aves, bovine, equine, avian, ovine, caprine, lagomorpha, and swine.
  • fish includes, but is not limited to, chondrichthyes, osteichthyes and agnatha.
  • mutant interferons includes mutant interferon polypeptides and mutant interferon polynucleotides.
  • mutant interferon refers to an interferon polypeptide or polynucleotide that is not naturally occurring. Mutant interferons retain biological activity. In some embodiments, mutant interferons may have enhanced biological activity or activities. In some embodiments, mutant interferons may have some " decreased biological activity or activities.
  • Mutant interferon polypeptides include interferons with an amino acid mutation, substitution or deletion, or a truncated polypeptide, compared to a naturally occurring interferon.
  • Mutant interferon polynucleotides also include polynucleotide sequences with a nucleotide substitution or deletion that may or may not encode a mutant interferon polypeptide.
  • mutant interferon polynucleotides include polynucleotide sequences encoding mutant interferon polypeptides, but also include, for example, codon-optimized polynucleotide sequences that encode a naturally occurring interferon polypeptide or a mutant polypeptide.
  • mutant and variant are used interchangeably.
  • naturally occurring and
  • wild-type and wild type are used interchangeably. Therefore, a mutant interferon is neither naturally occurring nor a wild-type.
  • biological activity includes one or more of the functions or activities of a naturally occurring or wild-type polypeptide and/or polynucleotide interferon.
  • Interferon biological activities may include, but are not limited to, antiviral activity, antiproliferative activity, induction of interferon gamma in NK 92 cells, and major histocompatibility complex (MHC) class I antigen expression induction activity.
  • MHC major histocompatibility complex
  • isolated refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule.
  • isolated also refers to a nucleic acid or peptide, polypeptide or protein that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • high stringent hybridization conditions is well understood in the art to encompass conditions of hybridization that allow hybridization of structurally related, but not structurally dissimilar, nucleic acids.
  • stringent is a term of art which is understood by the skilled artisan to describe any of a number of alternative hybridization and wash conditions which allow annealing of only highly complementary nucleic acids.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”.
  • antibody as it is used herein with respect to the invention, includes an isolated, recombinant or synthetic antibody, antibody conjugate or antibody derivative.
  • the term “antibody” is often intended to include an antibody fragment, including an antigen-binding fragment, unless otherwise indicated or understood by context.
  • An antigen-binding fragment competes with the intact antibody for specific binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)).
  • Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • antigen-binding fragments include Fab, F(ab)2, Fab', F(ab')2, F(ab')3, Fd, Fv, domain antibodies (dAb), other monovalent and divalent fragments, complementarity determining region (CDR) fragments, single- chain antibodies (e.g., scFv, scFab, and scFab ⁇ C), chimeric antibodies, diabodies, triabodies, minibodies, nanobodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide; and fusions and derivatives of the foregoing. See, e.g., Holliger and Hudson, Nature Biotechnology 23: 1126-1 136 (2005) and Hust et al., BMC Biotech 7: 14 (2007).
  • biologically acceptable medium includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. [0030
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.
  • treatment is intended to encompass also prophylaxis, therapy, cure, and prevention of spread of infection.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, the urinary bladder or other compartment of the body, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • therapeutically effective amount means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal and thereby blocking the biological consequences of that pathway in the treated cells, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agonists from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • a "pharmaceutical salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention.
  • Liposomes refer to lipid vesicles having an outer lipid shell, typically formed on one or more lipid bilayers, encapsulating an aqueous interior.
  • vesicle-forming lipid refers to any amphipathic lipid having hydrophobic and polar head group moieties and which by itself can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids.
  • microparticles includes microspheres (uniform spheres), microcapsules (having a core and an outer layer of polymer), and particles of irregular shape.
  • chemical derivatives thereof refer to substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art.
  • the present invention provides nucleic acids comprising a polynucleotide encoding an interferon polypeptide including an amino acid sequence shown in at least one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, or a fragment thereof.
  • one aspect of the invention provides a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; (b) a nucleotide sequence comprising one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19; (c) a nucleotide sequence encoding a biologically active interferon fragment of a polypeptide of (a) or (b) above; and (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b) or (c) above.
  • the fragments consist essentially of biologically active fragments of the interferon polypeptides.
  • Further embodiments of the invention include (1) a polynucleotide comprising a nucleotide sequence with at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to any of the nucleotide sequences in (a), (b), (c) or (d), above; (2) a polynucleotide that differs from any of the nucleotide sequences in (a), (b), (c) or (d), above, by 1 nucleotide, by 2 nucleotides, by 3 nucleotides, by 4 nucleotides, by 5 nucle
  • stringent hybridization conditions are selected to be about 5-10 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 0 C for short probes (e.g.
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In other embodiments, less stringent hybridization conditions may be used; for example, moderate or low stringency conditions may be used, and are well known in the art.
  • Exemplary high stringent hybridization conditions are equivalent to about 20-27 0 C below the melting temperature (T m ) of the DNA duplex formed in about 1 M salt.
  • T m melting temperature
  • high stringency refers to hybridization and/or washing conditions at 68°C in 0.2 x SSC, at 42 0 C in 50 % formamide, 4 x SSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
  • Suitable hybridization conditions for oligonucleotides e.g., for
  • oligonucleotide probes or primers are typically somewhat different than for full- length nucleic acids (e.g., full-length cDNA), because of the oligonucleotides' lower T m . Because the T m of oligonucleotides will depend on the length of the
  • suitable hybridization temperatures will vary depending upon the oligonucleotide molecules used. Exemplary temperatures may be 37 0 C (for 14-base oligonucleotides), 48 0 C (for 17-base oligonucleotides), 55 0 C (for 20-base oligonucleotides) and 60 0 C (for 23-base oligonucleotides).
  • Exemplary suitable hybridization conditions for oligonucleotides include washing in 6 x SSC, 0.05 % sodium pyrophosphate, or other conditions that afford equivalent levels of hybridization.
  • polypeptide, peptide and protein fragments of the invention may be biologically active fragments and may be of any length less than the full lengths of the sequences described in the sequence identifiers provided herein.
  • a fragment is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, or 170 amino acids in length.
  • nucleic acid fragments include those which may be useful as diagnostic probes and primers as discussed herein.
  • sequences may include those which specifically identify the interferon nucleotide sequences described herein, and they may be of any length. For example, they include those of at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably, at least about 40 nucleotides in length.
  • fragments such as those from 50-300 nucleotides in length are also useful according to the present invention as are fragments corresponding to at least one of the nucleotide sequences shown in at least one of SEQ ID NOS provided herein.
  • fragments at least 20 nucleotides in length for example, is intended fragments which include 20 or more contiguous bases from at least one of the nucleotide sequences as shown in at least one of SEQ ID NOS provided herein.
  • Fragments of 50-300 nucleotides in length include, for example, those which are about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 bases in length.
  • a polynucleotide that hybridizes to a portion of a polynucleotide includes a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably about 30-70 nucleotides, including any length in between (e.g. 50 bases), of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • the present invention also provides nucleic acids comprising a
  • polynucleotide encoding at least a portion of a human interferon polypeptide including an amino acid sequence shown in at least one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20, or a biologically active fragment thereof.
  • a polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; (b) a nucleotide sequence comprising one of SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 17, and 19; (c) a nucleotide sequence encoding a biologically active interferon fragment of a polypeptide of (a) or (b) above; and (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b) or (c) above.
  • the fragments consist essentially of biologically active fragments of the interferon polypeptides.
  • Further embodiments of the invention include (1) a polynucleotide comprising a nucleotide sequence with at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to any of the nucleotide sequences in (a), (b), (c) or (d), above; (2) a polynucleotide that differs from any of the nucleotide sequences in (a), (b), (c) or (d), above, by 1 nucleotide, by 2 nucleotides, by 3 nucleotides, by 4 nucleotides, by 5 nucle
  • any of the nucleic acids of the present invention which encode interferon polypeptides may include, but are not limited to, those encoding the amino acid sequence of the complete polypeptide, by itself; and the coding sequence for the complete polypeptide and additional sequences, such as those encoding an added secretory leader sequence, such as a pre-, or pro- or prepro-protein sequence.
  • nucleic acids described herein further comprising additional, non-coding sequences, including, for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example-ribosome binding and stability of mRNA; and an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • additional, non-coding sequences including, for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example-ribosome binding and stability of mRNA
  • additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif. 91311), among others, many of which are commercially available.
  • hexa- histidine as described by Gentz et al. provides for convenient purification of the fusion protein (Gentz et al. (1989) Proc. Natl. Acad. Sci.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. (1984) Cell 37: 767).
  • Other examples of fusion tags include the following: (1) MBP tag is a portion of maltose binding protein (vectors available from RocheTM (Example vector designation: pIVEX MBP), New England BiologicalTM (Example vector designation: pMAL-p2X); (2) HA tag is a portion of the hemagglutinin of human influenza virus (vectors available from RocheTM (Example vector designation: pIVEX HA-tag), BD BioscienccsTM (pCMV-HA)); (3) FLAG is a particular 8 amino acid epitope (vector from Sigma Chemical Co.TM (Example vector designation: gWiz/GFP); (4) CBP tag is a portion of calmodulin binding protein (vector available from Stratagene
  • Othcr common epitope tags for creating fusion proteins c-myc, GST, AUl, AU5, DDDDK, E epitope, E2 tag, Glu-Glu, Sl, KT-3, T7 epitope tag, V5 epitope tag, VSV-G, BFP, CFY, YFP.
  • other such fusion proteins include an interferon fused to Fc at the N- or C-terminus.
  • the present invention also provides recombinant vectors, which comprise the nucleic acids of the present invention, and host cells containing the recombinant vectors, as well as methods of making such vectors and host cells and for using them for production of interferon polypeptides or peptides by recombinant techniques.
  • 00541 The invention further provides an interferon polypeptide comprising an amino acid sequence selected from: (a) the amino acid sequence of an interferon polypeptide comprising an acid sequence shown in at least one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and (b) the amino acid sequence of a biologically active interferon fragment of a polypeptide of (a).
  • the fragments consist essentially of biologically active fragments of the interferon polypeptides.
  • Further embodiments of the invention include (1) biologically active polypeptides having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to any of the polypeptide sequences in (a) or (b) above; and (2) an amino acid sequence that differs from any of the amino acid sequences in (a) or (b), above, by 1 amino acid, by 2 amino acids, by 3 amino acids, by 4 amino acids, by 5 amino acids, by 6 amino acids, by 7 amino acids, by 8 amino acids, by 9 amino acids, by 10 amino acids, by 11 amino acids, by 12 amino acids, by 13 amino acids, by 14 amino acids
  • An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of an epitope-bearing portion of an interferon polypeptide having an amino acid sequence described herein.
  • Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of an interferon polypeptide of the invention include portions of such polypeptides with at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14 or at least 15, or more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above are included in the invention.
  • the invention further provides a human interferon polypeptide comprising an amino acid sequence selected from: (a) the amino acid sequence of an interferon polypeptide comprising an amino acid sequence shown in at least one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20; and (b) the amino acid sequence of a biologically active interferon fragment of a polypeptide of (a).
  • the fragments consist essentially of biologically active fragments of the interferon polypeptides.
  • inventions include (1) biologically active polypeptides having at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to any of the polypeptide sequences in (a) or (b) above; and (2) an amino acid sequence that differs from any of the amino acid sequences in (a) or (b), above, by 1 amino acid, by 2 amino acids, by 3 amino acids, by 4 amino acids, by 5 amino acids, by 6 amino acids, by 7 amino acids, by 8 amino acids, by 9 amino acids, by 10 amino acids, by 11 amino acids, by 12 amino acids, by 13 amino acids, by 14 amino acids, by 15 amino acids, by 16 amino acids, by 17 amino acids, by 18 amino acids, by 19 amino acids
  • each of these sequences and/or fragments is biologically active.
  • Another embodiment of the invention provides mutant interferons.
  • mutant interferon polypeptides of the invention differ from naturally occurring interferon polypeptides by 1 amino acid, by 2 amino acids, by 3 amino acids, by 4 amino acids, by 5 amino acids, by 6 amino acids, by 7 amino acids, by 8 amino acids, by 9 amino acids or by 10 amino acids.
  • mutant interferon polypeptides of the invention differ from naturally occurring interferon polypeptides by 15 or fewer amino acids, by 20 or fewer amino acids or by 25 or fewer amino acids.
  • each of these sequences and/or fragments is biologically active.
  • mutant interferon polypeptides of the invention share at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity with any of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20. According to the invention, each of these sequences and/or fragments is biologically active.
  • mutant interferon polynucleotides of the invention differ from naturally occurring interferon polynucleotides by 1 nucleotide, by 2 nucleotides, by 3 nucleotides, by 4 nucleotides, by 5 nucleotides, by 6 nucleotides, by 7 nucleotides, by 8 nucleotides, by 9 nucleotides, by 10 nucleotides, by 11 nucleotides, by 12 nucleotides, by 13 nucleotides, by 14 nucleotides, by 15 nucleotides, by 16 nucleotides, by 17 nucleotides, by 18 nucleotides, by 19 nucleotides, by 20 nucleotides, by 21 nucleotides, by 22 nucleotides, by 23 nucleotides, by 24 nucleotides, by 25 nucleotides, by 26 nucleotides, by 27 nucleotides, by 28 nucleotides, by 10
  • mutant interferon polypeptides of the invention differ from naturally occurring interferon polypeptides by 35 or fewer nucleotides, by 40 or fewer nucleotides or by 45 or fewer nucleotides. According to the invention, each of these sequences and/or fragments is biologically active.
  • mutant interferon polynucleotides of the invention share at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity with any of SEQ ID NOS: 1, 3, 5, 7, 9, 11 , 13, 15, 17, or 19. According to the invention, each of these sequences and/or fragments is biologically active.
  • mutant interferon polynucleotides include the backtranslated versions of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19.
  • mutant interferon polynucleotides include the reverse- backtranslated versions of SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, 17, or 19.
  • mutant interferon polypeptides of the invention may act as agonist or antagonists. While not wishing to be bound to any particular theory, it is well known that mutant forms of protein signaling factors are capable of binding to the appropriate receptor and yet not capable of activating the receptor. Such mutant proteins act as antagonists by displacing the wild-type proteins and blocking the normal receptor activation. Additionally, it is well known that one or more amino acid substitutions can be made to many proteins in order to enhance their activity in comparison to wild type forms of the protein. Such agonists may have, for example, increased half-life, binding affinity, stability, or activity in comparison to the wild type protein. There are many well known methods for obtaining mutants (or variants) with a desired activity.
  • the invention contemplates using interferon polypeptides generated by combinatorial mutagenesis.
  • Such methods are convenient for generating both point and truncation mutants, and can be especially useful for identifying potential variant sequences (e.g., homologs) that are functional in a given assay.
  • the purpose of screening such combinatorial libraries is to generate, for example, interferon variants or homologs that can act as either agonists or antagonists.
  • combinatorially derived variants can be generated to havc an increased potency relative to a naturally occurring form of the protein.
  • interferon variants can be generated by the present combinatorial approach to act as antagonists, in that they are able to mimic, for example, binding to other extracellular matrix components (such as receptors), yet not induce any biological response, thereby inhibiting the action of interferon polypeptides or interferon agonists.
  • manipulation of certain domains of interferon by the present method can provide domains more suitable for use in fi ⁇ sion or chimeric proteins, for example, domains demonstrated to have specific useful properties.
  • PCT publication WO92/ 15679 illustrate specific techniques which one skilled in the art could utilize to generate libraries of variants which can be rapidly screened to identify variants/fragments which possess a particular activity. These techniques are exemplary of the art and demonstrate that large libraries of related variants/truncants can be generated and assayed to isolate particular variants without undue experimentation. Gustin et al. (1993) Virology 193:653, and Bass et al. (1990) Proteins: Structure, Function and Genetics 8:309-314, also describe other exemplary techniques from the art which can be adapted as a means for generating mutagenic variants of the interferon polypeptides of the invention.
  • Table 1 illustrates the common alleles of the human interferon ⁇ family of genes/proteins and was constructed based on Pestka, S. (1983) Arch Biochem Biophys 221 : 1-37; Diaz, M.O., Pomykala, H.M., Bohlander, S.K., Maltepe, E., Malik, K., Brownstein, B., and Olopade, O.I. (1994) Genomics 22:540-52; and Pestka, S. (1986) Meth.
  • Enzymol 1 19:3-14 and reviewed in Krause, CD., Lunn, C.A., Izotova, L.S., Mirochnitchenko, O., Kotenko, S.V., Lundell, D.J., Narula, S. K., and Pestka, S. (2000) J Biol Chem. 275:22995-3004. Tabic 1
  • a preferred method for determining the best overall match between a query sequence and a subject sequence can be determined using the FASTDB computer program based on the algorithm of
  • Protein and/or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN,
  • BLAST Basic Local Alignment Search Tool
  • BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database
  • BLASTX compares the six- frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database
  • TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands).
  • TBLASTX compares the six-frame translations of a nucleotide query sequence against the six- frame translations of a nucleotide sequence database.
  • the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
  • High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.
  • the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445; Henikoff and Henikoff, 1993, Proteins 17:49-61).
  • the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).
  • the BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably select those segments which satisfy a user- specified threshold of significance, such as a user-specified percent homology.
  • the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).
  • the parameters used with the above algorithms may be adapted depending on the sequence length and degree of homology studied. In some embodiments, the parameters may be the default parameters used by the algorithms in the absence of instructions from the user.
  • the FASTDB algorithm described in Brutlag et al. Comp. App. Biosci. 6:237-245, 1990, is used.
  • nucleic acids encoding proteins having at least 90%, at least 91% sequence homology, at least 92% sequence homology, at least 93% sequence homology, at least 94% sequence homology, at least 95% sequence homology, at least 96% sequence homology, at least 97% sequence homology, at least 98% sequence homology, at least 99% sequence homology, at least 99.25% sequence homology, at least 99.50% sequence homology, at least 99.75% sequence homology, or 100% sequence homology to a protein encoded by a nucleic acid.
  • the homology levels can be determined using the "default" opening penalty and the "default” gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoffet al., in: Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)).
  • a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoffet al., in: Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)).
  • the level of polypeptide homology may be determined using the FASTDB algorithm described by Brutlag et al. Comp. App. Biosci. 6:237-245, 1990.
  • the invention also encompasses sequences having a lower degree of identity than that described herein but having sufficient similarity so as to perform one or more of the same functions. Similarity is determined by conserved amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu, and He, interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and GIu, substitution between the amide residues Asn and GIn, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
  • Guidance concerning which amino acid changes are likely to be phenotypically silent is found in Bowie et al., Science 247:1306-1310 (1990).
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J MoI. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web site at GCG.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available on the world wide web site at GCG.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4: 1 1-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. MoI. Biol. 215:403-10 (1990)).
  • Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used.
  • the invention provides an antibody that binds specifically to an interferon polypeptide of the invention.
  • the invention further provides methods for isolating antibodies that bind specifically to an interferon polypeptide of the invention. Such antibodies are useful therapeutically as described below.
  • the antibodies of the invention include whole antibodies and any antigen- binding fragments thereof, antibody derivatives or variants that may contain one or more modifications (e.g., an amino acid insertion, deletion, substitution, a post- translational modification or lack thereof, etc.), including antibody conjugates (i.e., antibody or antigen-binding fragment thereof conjugated to or associated with a functional moiety).
  • the antibody derivatives, including antibody conjugates may be based on or may comprise an antigen-binding fragment of the invention that specifically binds an epitope or polypeptide of the invention.
  • the aforementioned antibody embodiments may be murine, mouse, rat, hamster, goat, camel, rabbit, chimeric, humanized, or fully human antibodies, fragments, derivatives, or conjugates. It is understood that in certain aspects of the invention, the term "antibody” may exclude one or more of the antibody embodiments recited above; such conditions will be evident to the skilled artisan.
  • An antibody may be a monoclonal antibody, a polyclonal antibody, a murine antibody, mouse antibody, rat antibody, hamster antibody, goat antibody, camel antibody, rabbit antibody, a chimeric antibody, a primatized antibody, a humanized antibody, a (fully) human antibody, a multimeric antibody, a heterodimeric antibody, a hemidimeric antibody, a bi-, tri-, or tetravalent antibody, a bispecific antibody, a single chain antibody (e.g., scFv, scFab, and scFab ⁇ C), Bis-scFv, a diabody, triabody or tetrabody, nanobody, minibody, single domain antibodies, and modified Fab fragments.
  • a single chain antibody e.g., scFv, scFab, and scFab ⁇ C
  • Bis-scFv a diabody, triabody or tetrabody, nanobody, minibody, single domain antibodies
  • the antibody comprises only a single variable immunoglobulin domain.
  • monovalent antibodies include antibodies that comprise only one immunoglobulin variable domain (i.e., a single light or heavy variable chain) and that specifically bind to an epitope or polypeptide of the invention.
  • antibodies of the invention may be monovalent, divalent, or multivalent for an epitope or polypeptide of the invention.
  • Antibody fragments include, for example, an Fab fragment, an F(ab)2 fragment, an Fab' fragment, an F(ab')2 fragment, an F(ab')3, fragment, a single chain F(v) fragment or an F(v) fragment and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9): 1 126-1136). Antibody fragments of the invention are described in more detail below.
  • the antibody molecules of the invention can be of any class (e.g. IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecule.
  • the constant region domains of the antibody if present, may be selected having regard to the proposed function of the antibody molecule.
  • the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains.
  • human IgG constant region domains may be used, especially IgGl, IgG2, IgG3, and IgG4.
  • IgG2 and IgG4 isotypes may be used in certain embodiments where the antibody molecule is intended for therapeutic uses for which reduced or eliminated antibody effector functions are desired.
  • IgGl and IgG3 isotypes may be used when the antibody molecule is intended for therapeutic purposes for which antibody effector functions are required.
  • one or more of the CDRs of a polypeptide of the invention may be incorporated into one or more immunoglobulin domains, universal frameworks, protein scaffolds or other biocompatible framework structures based on protein scaffolds or skeletons other than immunoglobulin domains (Nygren & Uhlen, 1997, Curr. Opin. Struct. Biol. 7:463-469; Saragovi et al, 1992, Bio/Technology 10:773-779; Skerra, 2000, J. MoI. Recognition 13: 167-187).
  • the CDRs of an antibody are incorporated into a universal framework (i.e., a framework which can be used to create the full variability of functions, specificities, or properties which are originally sustained by a large collection of different frameworks, see U.S. 6,300,064).
  • a universal framework i.e., a framework which can be used to create the full variability of functions, specificities, or properties which are originally sustained by a large collection of different frameworks, see U.S. 6,300,064
  • alternative scaffolds see, for example, Binz et al. 2005 Nat Biotech 23: 1257-1268 and Hosse et al. 2006 Protein Science 15: 14-27 may be used.
  • the antibody is a synthetic antibody or a recombinant antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage.
  • An antibody of the invention may also include an antibody that has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology that is available and well known in the art.
  • the invention includes antibodies that can act as interferon antagonists.
  • Antibodies can have extraordinary affinity and specificity for particular epitopes. The binding of an antibody to its epitope on a protein may antagonize the function of that protein by competitively or non-competitively inhibiting the interaction of that protein with other proteins necessary for proper function.
  • Antibodies with interferon antagonist activity can be identified in much the same way as other interferon antagonists.
  • candidate antibodies can be administered to cells expressing a reporter gene, and antibodies that cause decreased reporter gene expression are antagonists.
  • antibodies of the invention can be single chain antibodies (scFv), comprising variable antigen binding domains linked by a polypeptide linker.
  • Single chain antibodies are expressed as a single polypeptide chain and can be expressed in bacteria and as part of a phage display library. In this way, phage that express the appropriate scFv will have interferon antagonist activity.
  • the nucleic acid encoding the single chain antibody can then be recovered from the phage and used to produce large quantities of the scFv. Construction and screening of scFv librarics is extensively described in various publications (U.S. Patents 5,258,498; 5,482,858; 5,091,513; 4,946,778; 5,969,108; 5,871,907; 5,223,409; 5,225,539).
  • the invention further provides compositions comprising any of the interferon polynucleotides or interferon polypeptides, described herein, for administration to cells in vitro, to cells ex vivo, and to cells in vivo, or to a multicellular organism.
  • the compositions comprise an interferon polynucleotide for expression of an interferon polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with loss or lack of activity of an endogenous interferon.
  • the invention contemplates methods of treatment using interferon polynucleotides and pharmaceutical compositions comprising interferon
  • Interferon polynucleotides may be delivered to cells using any technique known in the art including, for example, viral packaging, lipofection or microinjection.
  • Nucleic acids may also be carried to cells via polymeric
  • vectors comprising interferon polynucleotides Any methods for controlled expression of vectors comprising interferon polynucleotides may be used. Methods for regulating expression vectors are well known in the art. For example, vectors used to deliver interferon polynucleotides may be cell type-specific, cell status-specific or inducible.
  • compositions comprising interferon polypeptides of the invention and methods which may be employed, for instance, to treat or prevent immune system-related disorders such as viral infection, parasitic infection, bacterial infection, cancer, autoimmune disease, multiple sclerosis, lymphoma and allergy. Methods of treating individuals or subjects in need of interferon polypeptides are also provided.
  • the subject pharmaceutical composition is a veterinary composition for administration to a non-human animal, preferably a non-human primate.
  • Exemplary conditions which may be treated with an interferon include but are not limited to viral infections.
  • treatment with interferon may be used to treat conditions which would benefit from inhibiting the replication of intcrfcron-sensitive viruses.
  • Viral infections which may be treated in accordance with the invention include severe acute respiratory syndrome-associated
  • coronavirus coronavirus
  • influenza smallpox virus
  • cowpox virus monkeypox virus
  • encephalitis-causing viruses hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV),
  • EBV Epstein-Barr virus
  • CMV cytomegalovirus
  • HHV-6 human herpes virus type 6
  • vaccinia virus vaccinia virus
  • rhinovirus arterivirus
  • filovirus picornavirus
  • reovirus rotavirus
  • rabies papilloma virus
  • retroviruses including human immunodeficiency viruses (HIV), human T lymphotropic virus- type 1 and 2 (HTLV-I /-2), papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echovirus,
  • enterovirus enterovirus
  • cardiovirus togavirus
  • rhabdovirus togavirus
  • bunyavirus bunyavirus
  • arenavirus
  • the method of the invention may also be used to modify various immune responses.
  • the interferon polypeptides described herein may be used for treating viral infections caused by viruses such as those described herein. These interferon polypeptides may also used in a prophylactic manner, such as by preventing the infection or preventing the subject from exhibiting symptoms associated with the infection. Subjects who may benefit from such preventive treatment include those with an elevated risk of being infected, such as subjects who have become exposed to the virus or to individuals who have been infected by or exposed to the virus.
  • an interferon may be used as an anti- viral agent.
  • Interferons have been used clinically for the treatment of acquired immune disorders, viral hepatitis including chronic hepatitis B, hepatitis C, hepatitis D, papilloma viruses, herpes, viral encephalitis, and in the prophylaxis of rhinitis and respiratory infections.
  • an interferon of the invention may be used as an anti-parasitic agent.
  • the interferons may be used, for example, for treating
  • an interferon of the- invention may be used as an anti-bacterial agent, interferons have been used clinically for anti-bacterial therapy.
  • interferons may be used in the treatment of multidrug-resistant pulmonary tuberculosis.
  • an interferon of the invention may be used as part of an immunotherapy protocol.
  • the interferons of the present invention may be used clinically for immunotherapy or more particularly, for example, to prevent graft vs. host rejection, or to curtail the progression of autoimmune diseases, such as arthritis, multiple sclerosis, or diabetes.
  • an interferon of the invention may be used as part of a program for treating allergies.
  • interferons can be used as vaccine adjuvants, interferons may be used as an adjuvant or coadjuvant to enhance or stimulate the immune response in cases of prophylactic or therapeutic vaccination.
  • the interferons of the invention may be used for the treatment of primates as part of veterinary protocols.
  • 01011 In addition to the treatment of animals in general, the interferons of the invention may be used for the treatment of fish.
  • An interferon of the invention may be used for treating cats.
  • an interferon of the invention may be used to treat dogs or other household pets, (de Mari K, Maynard L, Eun HM, Lebreux B. Vet Rec. (2003) 152: 105-8).
  • an interferon of the invention may be used to treat farm animals.
  • an interferon of the invention may be used to treat fish.
  • an interferon of the invention may be used to treat humans.
  • This invention further provides a method of treating a subject infected with a virus selected from the group consisting of severe acute respiratory syndrome- associated coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruses, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picomavirus, reovirus, rotavirus, rabies, papilloma virus, retroviruses including human immunodeficiency viruses (HIV), human T lymphotropic virus-type 1 and 2
  • SARS severe acute respiratory syndrome- associated coronavirus
  • HTLV-I /-2 papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echovirus, enterovirus, cardio virus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus, comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of virus particles in the subject, thereby treating the subject.
  • This invention contemplates any of the treatment methods described herein also as methods for preventing the subject from becoming afflicted or infected, or as methods of reducing the subject's risk of a affliction or infection, or as protecting the subject against disorders/conditions related to a particular virus, or as preventing the subject from exhibiting symptoms associated with a viral infection.
  • the above methods for treating subject infected with a virus may also be used to prevent the subject from becoming infected with the virus, or to reduce the subject risk of viral infection.
  • This invention provides a method of reducing a subject's risk of viral infection comprising administering to the subject an interferon polypeptide. In one embodiment, this method comprises preventing the subject from being infected with the virus. In one embodiment, this method comprises preventing the subject from exhibiting symptoms associated with a viral infection. In one embodiment, this method comprises protecting the subject against disorders/conditions related to a particular virus. This protection may be conferred by preventing or lessening the severity of a disorder/condition resulting from the infection. In another
  • the protection may also be conferred by reducing the spread of infection to others by lessening the severity of a disorder/condition resulting from the infection in the patient.
  • the prevention or reduction of risk is effected by causing the subject's cells to become less susceptible to infection.
  • the viral infections include but are not limited to those caused by severe acute respiratory syndrome-associated coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruses, hepatitis A, hepatitis B, hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, rotavirus, rabies, papilloma virus, retroviruses including human immunodeficiency viruses (HIV), human T lymphotropic virus-type 1 and 2 (HTLV-I /-2), papovavirus, herpesvirus, poxvirus, hepadnavirus,
  • orthomyxovirus orthomyxovirus, echovirus, enterovirus, cardiovirus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus.
  • This invention provides a method of treating a subject afflicted with influenza (orthomyxovirus), comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of influenza virus particles in the subject, wherein the interferon polypeptide comprises an amino acid sequence that has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to at least one of the SEQ ID NOS described herein, thereby treating the subject.
  • each of these sequences and/or fragments is biologically active.
  • This invention provides a method of treating a subject afflicted with Hepatitis C, comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of Hepatitis C virus particles in the subject, wherein the interferon polypeptide comprises an amino acid sequence that has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to at least one of the SEQ ID NOS described herein, thereby treating the subject.
  • each of these sequences and/or fragments is biologically active.
  • This invention provides a method of treating a subject afflicted with rhinovirus, coronavirus, or arenavirus, comprising administering to the subject an amount of an interferon polypeptide that is effective to reduce the concentration of rhinovinis, coronavirus, or arenavirus virus particles in the subject, wherein the interferon polypeptide comprises an amino acid sequence that has at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, at least 99.25% sequence identity, at least 99.50% sequence identity, at least 99.75% sequence identity, or 100% sequence identity to at least one of the SEQ ID NOS described herein, thereby treating the subject.
  • each of these sequences and/or fragments is biologically active.
  • interferon polypeptides referred to herein include but are not limited to IFN- ⁇ polypeptides, IFN- ⁇ polypeptides, IFN- ⁇ polypeptides, and IFN- ⁇ polypeptides (Zoon KC: Human Interferons: Structure and Function, p. 1-12. In: Interferon 8.
  • Pestka, S. Biopolymers 2000; 55(4):254-287; Biopolymers 2000; 55(4):254-287; Pestka, S., Methods in Enzymology, 78, 1981; Pestka, S., Methods in Enzymology, 79, 1981; Pestka, S., Methods in Enzymology, 119, 1986; Pestka, S., Langer; J. A., Zoon, K.C., Samuel, CE. Ann Rev Biochem 1987, 56, 727-777).
  • This invention also provides a method of preventing a subject from becoming afflicted with a syndrome caused by a virus selected from the group consisting of severe acute respiratory syndrome-associated coronavirus (SARS), coronavirus, influenza, smallpox virus, cowpox virus, monkeypox virus, encephalitis-causing viruses, hepatitis A, hepatitis B, hepatitis C, other non-A/non- B hepatitis, herpes virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virus type 6 (HHV-6), West Nile virus, vaccinia virus, respiratory syncytial virus, rhinovirus, arterivirus, filovirus, picornavirus, reovirus, rotavirus, rabies, papilloma virus, retroviruses including human
  • SARS severe acute respiratory syndrome-associated coronavirus
  • coronavirus coronavirus
  • influenza smallp
  • immunodeficiency viruses HAV
  • HTLV- 1/-2 papovavirus, herpesvirus, poxvirus, hepadnavirus, astrovirus, coxsackie virus, paramyxovirus, orthomyxovirus, echovirus, enterovirus, cardiovirus, togavirus, rhabdovirus, bunyavirus, arenavirus, bornavirus, adenovirus, parvovirus, and flavivirus, comprising administering to the subject an amount of an interferon polypeptide.
  • This invention provides a method of preventing a subject from becoming afflicted with influenza, comprising administering to the subject an amount of an interferon polypeptide described herein.
  • This invention provides a method of reducing the risk of a subject from becoming infected with influenza, comprising administering to the subject an amount of an interferon polypeptide described herein.
  • the subject is a human being.
  • the subjects include but are not limited to dogs, cats, fish, monkeys, and farm animals.
  • This invention provides a method of preventing a subject from becoming afflicted with Hepatitis C, comprising administering to the subject an amount of an interferon polypeptide described herein.
  • This invention provides a method of reducing the risk of a subject from becoming infected with Hepatitis C, comprising administering to the subject an amount of an interferon polypeptide described herein.
  • the subject is a human being.
  • the subjects include but are not limited to dogs, cats, fish, monkeys, and farm animals.
  • This invention provides a method of preventing a subject from becoming afflicted with rhino virus, coronavirus, or arenavirus comprising administering to the subject an amount of an interferon polypeptide described herein.
  • This invention provides a method of reducing the risk of a subject from becoming infected with rhinovirus, coronavirus, or arenavirus comprising administering to the subject an amount of an interferon polypeptide described herein.
  • the subject is a human being.
  • the subjects include but are not limited to dogs, cats, fish, monkeys, and farm animals.
  • This invention provides the use of an interferon polypeptide of the invention for the preparation of a medicament for any of the methods of treatment, prevention, prophylaxis, and reduction of risk described herein.
  • This invention provides a pharmaceutical package comprising an interferon composition with instructions for administering the composition to a subject.
  • the instructions may include written and/or pictorial instructions.
  • the instructions may bc for any of the methods or treatment, prevention, prophylaxis, and reduction of risk described herein.
  • the subject invention also contemplates functional antagonists, e.g., wherein one or more amino acid residues are different from the naturally occurring interferon, which inhibit one or more biological activities of the naturally occurring interferon.
  • Such antagonists can be used to treat disorders resulting from aberrant overexpression or other activation of an endogenous interferon.
  • the functional antagonists may be formulated in a pharmaceutical preparation.
  • the present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a biological activity of an interferon polypeptide, which involves contacting a receptor which is enhanced by an interferon polypeptide with the candidate compound in the presence of an interferon polypeptide, assaying, for example, anti- viral activity in the presence of the candidate compound and an interferon polypeptide, and comparing the activity to a standard level of activity, the standard being assayed when contact is made between the receptor and interferon in the absence of the candidate compound.
  • an increase in activity over the standard indicates that the candidate compound is an agonist of interferon activity and a decrease in activity compared to the standard indicates that the compound is an antagonist of interferon activity.
  • An additional aspect of the invention is related to a method for treating an animal in need of an increased level of interferon activity in the body comprising administering to such an animal a composition comprising a therapeutically effective amount of an isolated interferon polypeptide of the invention or an agonist thereof.
  • a still further aspect of the invention is a method for treating an animal in need of a decreased level of interferon activity in the body comprising, administering to such an animal a composition comprising a therapeutically effective amount of an interferon antagonist.
  • Preferred antagonists for use in the present invention are interfcron-specific antibodies.
  • administration of the described dosages may be every other day, but is preferably once or twice a week. Doses may be administered over at least a 24 week period by injection. [0121) Administration of the dose can be intravenous, subcutaneous, intramuscular, or any other acceptable systemic method. Based on the judgment of the attending clinician, the amount of drug administered and the treatment regimen used will, of course, be dependent on the age, sex and medical history of the patient being treated, the neutrophil count (e.g. the severity of the neutropenia), the severity of the specific disease condition and the tolerance of the patient to the treatment as evidenced by local toxicity and by systemic side-effects. Dosage amount and frequency may be determined during initial screenings of neutrophil count.
  • formulations can be also prepared using the subject interferon compositions of the present invention.
  • the formulations comprise a therapeutically effective amount of an interferon polypeptide together with pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers for example, adjuvants, diluents, preservatives and/or solubilizers, if needed, may be used in the practice of the invention.
  • compositions of interferon including those of the present invention may include diluents of various buffers (e.g., Tris-HCl, acetate, phosphate) having a range of pH and ionic strength, carriers (e.g., human serum albumin), solubilizers (e.g., Polyoxyethylene Sorbitan or TWEENTM polysorbate), and preservatives (e.g., thimerosal, benzyl alcohol). See, for example, U.S. Pat. No. 4,496,537.
  • buffers e.g., Tris-HCl, acetate, phosphate
  • carriers e.g., human serum albumin
  • solubilizers e.g., Polyoxyethylene Sorbitan or TWEENTM polysorbate
  • preservatives e.g., thimerosal, benzyl alcohol
  • the therapeutic amount of the interferon composition administered to treat the conditions described above is based on the interferon activity of the composition. It is an amount that is effective to significantly affect a positive clinical response.
  • a positive clinical response may be indicated by a reduction in the concentration of virus particles in the subject, or more generally as a reduction in the symptoms of the infection.
  • a positive clinical response may be indicated, for example, by an absence of virus particles in the subject, by a reduction in the concentration of virus particles in the subject, or by maintaining the concentration of virus particles in the subject below the threshold concentration above which the subject exhibits the symptoms of the viral infection.
  • a prophylactic treatment includes preventing a subject from becoming afflicted with a disorder caused by a virus.
  • the clinical dose may cause some level of side effects in some patients
  • the maximal dose for mammals including humans is the highest dose that does not cause unmanageable clinically- important side effects.
  • clinically important side effects are those which would require cessation of therapy due to severe flu-like symptoms, central nervous system depression, severe gastrointestinal disorders, alopecia, severe pruritus or rash.
  • Substantial white and/or red blood cell and/or liver enzyme abnormalities or anemia- like conditions are also dose limiting.
  • interferon may vary somewhat depending upon the formulation selected. In general, however, the interferon composition is administered in amounts ranging from about 100,000 to about several million IU/m 2 per day, based on the mammal's condition. The range set forth above is illustrative and those skilled in the art will determine the optimal dosing of interferon selected based on clinical experience and the treatment indication.
  • compositions may be in the form of a solution, suspension, tablet, capsule, lyophilized powder or the like, prepared according to methods well known in the art. It is also contemplated that administration of such compositions will be chiefly by the parenteral route although oral or inhalation routes may also be used depending upon the needs of the artisan.
  • the present invention provides pharmaceutical preparations comprising interferons, interferon agonists or interferon antagonists (collectively referred to herein as the compounds of the invention).
  • the interferons, interferon agonists and/or interferon antagonists for use in the subject method may be conveniently formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • a biologically acceptable medium such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists.
  • compositions of the present invention can also include veterinary compositions, e.g., pharmaceutical preparations of the compositions of the present invention suitable for veterinary uses, e.g., for the treatment of livestock, fish, non-human primate, or domestic animals, e.g., dogs and cats.
  • Rechargeable or biodegradable devices may also provide methods of introduction.
  • Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals.
  • a variety of biocompatible polymers including hydrogels, including both biodegradable and non-degradable polymers, can be used to form an implant for sustained release at a particular target site.
  • the preparations of the present invention may be administered to humans and other animals by any suitable route of administration, including orally, nasally (as by, for example, a spray), parenterally, topically (as by powders, ointments or drops, including buccally and sublingually), intrathecally/intracerebroventricularly (ICV), intracranially, directly into the central nervous system (intracavitary), intravaginally, intracisternally, or rectally.
  • the preparations are administered in forms suitable for each administration route.
  • the interferon is delivered directly to nasopharyngeal mucosa.
  • the interferon is delivered directly to the lung epithelium. Unlike using these tissues as a vehicle for systemic delivery, direct delivery to these cells may make them resistant to viruses. Moreover, it reduces the amount of systemic interferon so side effects will be minimal or eliminated.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms such as described below or by other conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including: the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof; the route of administration; the time of administration; the rate of excretion of the particular compound being employed; the duration of the treatment; other drugs, compounds and/or materials used in combination with the particular composition employed; the age, sex, weight, condition, general health and prior medical history of the patient being treated; and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and-prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the patient receiving this treatment may be any animal in need thereof or any animal that is the source of virus (animal vector), including: mammals such as equines, cattle, swine, rodents and sheep; poultry; and pets in general.
  • the patient is a primate (in particular, a human) or another mammal.
  • the compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable and/or sterile carriers and can also be administered in conjunction with other agents.
  • agents include antimicrobial agents such as penicillins, cephalosporins, aminoglycosides, and glycopeptides.
  • Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutic effects of the first administered one is not entirely disappeared when the subsequent is administered.
  • compositions of the present invention may be formulated for administration in any convenient way for use in human or veterinary medicine.
  • the compound included in the pharmaceutical preparation may be active itself, or may be a prodrug, e.g., capable of being converted to an active compound in a physiological setting.
  • compositions comprising a therapeutically effective amount of one or more of the compounds of the invention, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • pharmaceutically acceptable carriers additives
  • the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by
  • subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension
  • topical application for example, as a cream, ointment or spray applied to the skin
  • intravaginally or intrarectally for example, as a pessary, cream or foam.
  • the subject compounds may be simply dissolved or suspended in sterile water.
  • the pharmaceutical preparation is non-pyrogenic, i.e., does not elevate the body temperature of a patient.
  • Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, saf ⁇ lower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide
  • the compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, for example, liposomes, polymers, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • the subject interferons can be provided in formulations also including penetration enhancers, carrier compounds and/or transfection agents.
  • compositions of the invention also encompass any pharmaceutically acceptable salts, esters or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the compositions also include to pharmaceutically acceptable salts and other bioequivalents.
  • the formulations are as part of a "supramolecular complex.”
  • the interferon can be contacted with at least one polymer to form a composite and then the polymer of the composite treated under conditions sufficient to form a supramolecular complex containing the interferon and a multi-dimensional polymer network.
  • the polymer molecule may be linear or branched. Accordingly, a group of two or more polymer molecules may be linear, branched, or a mixture of linear and branched polymers.
  • the composite may be further modified with at least one ligand, e.g., to direct cellular uptake of the expression construct or otherwise effect tissue or cellular distribution in vivo of the exprcssion construct.
  • the composite may take any suitable form and, preferably, is in the form of particles.
  • the supramolecular complexes are aggregated into particles, for example, formulations of particles having an average diameter of between 20 and 5000 nanometer (nm). In another embodiment, the particles have an average diameter of between 20 and 200 nm. In another embodiment, the particles have an average diameter of between 2 and 10 microns. Use of a particle size of between 2 and 10 microns may be used for example, for delivery to the lung.
  • the interferons are provided in cationic, non-lipid vehicles and formulated to be used in aerosol delivery via the respiratory tract.
  • PEI poly(ethylenimine)
  • macromolecules such as dsRNA and dsRNA-encoding plasmids
  • PEI-nucleic acid formulations can also exhibit a high degree of specificity for the lungs.
  • the invention also contemplates the use of cyclodextrin-modified polymers, such as cyclodextrin- modified poly(ethylenimine).
  • the invention provides a composition including interferons that are encapsulated or otherwise associated with liposomes.
  • the liposomes are cationic liposomes composed of between about 20-80 mole percent of a cationic vesicle-forming lipid, with the remainder neutral vesicle-forming lipids and/or other components.
  • the lipid is a vesicle-forming lipid.
  • a preferred vesicle-forming lipid is a diacyl-chain lipid, such as a phospholipid, whose acyl chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • Neutral vesicle-forming lipids are those vesicle forming lipids which have no net charge or which may include a small percentage of lipids having a negative charge in the polar head group. Included in this class of lipids are the phospholipids, such as phosphatidylcholine (PC), phosphatidyl ethanolamine (PE),
  • PC phosphatidylcholine
  • PE phosphatidyl ethanolamine
  • lipids can be obtained commercially, or prepared according to published methods.
  • Other lipids that can be included in the invention ⁇ are glycolipids, such as cerebrosides and gangliosides.
  • the interferon- liposome complex includes liposomes having a surface coating of hydrophilic polymer chains, effective to extend the blood circulation time of the plasmid/liposome complexes.
  • Suitable hydrophilic polymers include cyclodextrin (CD), polyethylene glycol (PEG), polylactic acid, polyglycolic acid, polyvinyl-pyrrolid-one, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethyl-cellulose.
  • a preferred hydrophilic polymer chain is
  • polyethyleneglycol preferably as a PEG chain having a molecular weight between 500-10,000 Daltons, more preferably between 1 ,000-5,000 Daltons.
  • the hydrophilic polymer may have solubility in water and in a non-aqueous solvent, such as chloroform.
  • hydrophilic polymer can be stably coupled to the lipid, or coupled through an unstable linkage which allows the polymer-coated plasmid- liposome complexes to shed or "release" the hydrophilic polymer coating during circulation in the bloodstream or after localization at a target site.
  • Attachment of hydrophilic polymers, in particular polyethyleneglycol (PEG), to vesicle- forming lipids through a bond effective to release the polymer chains in response to a stimulus have been described, for example in WO 98/16202, WO 98/16201, which are hereby incorporated by reference, and by Kirpotin, D. et al. (FEBS Letters, 388:1 15-1 18 (1996).
  • the hydrophobic segment in the polymer-lipid conjugate is a hydrophobic polypeptide sequence.
  • the polypeptide sequence consists of about 5-80, more preferably 10-50, most preferably 20-30, non- polar and/or aliphatic/aromatic amino acid residues. These sequences are active in triggering fusion of certain enveloped viruses with host cells and include
  • parainfluenza viruses such as Sendai, Simian Virus-5 (SV5), measles virus,
  • Newcastle Disease Virus NDV
  • Respiratory Syncytial Virus RSV
  • Other examples include human retroviruses, such as Human Immunodeficiency Virus- 1 (HIV-I), the causative agent of AIDS, which infects cells by fusion of the virus envelope with the plasma membrane of the host cell. Fusion occurs at physiological (i.e., neutral) pH and is followed by injection of the viral genetic material
  • nucleocapsid into the cytoplasmic compartment of the host cell.
  • the polymeric complexes such as the
  • supramolecular complexes, and liposomes of the subject invention can be associated with one or more ligands effective to bind to specific cell surface proteins or matrix on the target cell, thereby facilitating sequestration of the complex to target cells, and in some instances, enhancing uptake of the interferon by the cell.
  • ligands suitable for use in targeting the supramolecular complexes and liposomes of the present invention to specific cell types are listed in Table 2 below.
  • the present invention also contemplates the derivatization of the subject polymeric and liposomal complexes with ligands that promote transcytosis of the complexes.
  • a polymeric complex such as a supramolecular complex
  • an internalizing peptide which drives the translocation of the complex across a cell membrane in order to facilitate intracellular localization of the interferon.
  • the internalizing peptide by itself, is capable of crossing a cellular membrane by, e.g., transcytosis, at a relatively high rate.
  • the internalizing peptide is conjugated, e.g., as covalent pendant group, to the polymer.
  • Pore-forming proteins or peptides may serve as internalizing peptides herein. Pore-forming proteins or peptides may be obtained or derived from, for example, C9 complement protein, cytolytic T-cell molecules or NK-cell molecules. These moieties are capable of forming ring-like structures in membranes, thereby allowing transport of attached complexes through the membrane and into the cell interior.
  • an internalizing peptide may be sufficient for translocation of the complexes across cell membranes.
  • translocation may be improved by attaching to the internalizing peptide a substrate for intracellular enzymes (i.e., an "accessory peptide").
  • an accessory peptide be attached to a portion(s) of the internalizing peptide that protrudes through the cell membrane to the cytoplasmic face.
  • the accessory peptide may be advantageously attached to one terminus of a translocating/internalizing moiety or anchoring peptide.
  • An accessory moiety of the present invention may contain one or more amino acid residues.
  • an accessory moiety may provide a substrate for cellular phosphorylation (for instance, the accessory peptide may contain a tyrosine residue).
  • the internalizing and accessory peptides can each, independently, be added to an interferon complex or liposome by chemical cross-linking or through non- covalent interaction (e.g., use of streptavidin-biotin conjugates, His6-Ni interactions, etc).
  • unstructured polypeptide linkers can be included between the peptide moieties and the polymeric complex or liposome.
  • Such internalizing and accessory peptides can be associated directly with an interferon, such as through a covalent linkage to a hydro xyl group on the backbone of the protein.
  • the linkage is susceptible to cleavage under physiological conditions, such as by exposure to esterases, or simple hydrolysis reactions.
  • Such compositions can be used alone or formulated in polymeric complexes or liposomes.
  • the respiratory tract includes the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli.
  • the upper and lower airways are called the conductive airways.
  • the terminal bronchioli then divide into respiratory bronchioli which then lead to the ultimate respiratory zone, the alveoli, or deep lung.
  • Administration by inhalation may be oral and/or nasal, intratracheal (trans- stomal or via tracheostomy tube), or via a breathing assistance device such as a respirator.
  • a breathing assistance device such as a respirator.
  • pharmaceutical devices for aerosol delivery include metered dose inhalers (MDIs), dry powder inhalers (DPIs), and air-jet nebulizers.
  • MDIs metered dose inhalers
  • DPIs dry powder inhalers
  • air-jet nebulizers Exemplary delivery systems by inhalation which can be readily adapted for delivery of the subject interferons are described in, for example, U.S. patents 5,756,353; 5,858,784; and PCT applications WO98/31346; WO98/10796; WOOO/27359; WOO 1/54664; WO02/060412.
  • alveolar macrophages are capable of phagocytosing particles soon after their deposition. Warheit et al. Microscopy Res. Tech., 26: 412-422 (1993); and Brain, J. D., "Physiology and Pathophysiology of Pulmonary Macrophages," in The
  • the aerosolized interferons are formulated as microparticles.
  • Microparticles having a diameter of between 0.5 and ten microns can penetrate the lungs, passing through most of the natural barriers. A diameter of less than ten microns is required to bypass the throat; a diameter of 0.5 microns or greater is required to avoid being exhaled.
  • the subject interferons are formulated in a supramolecular complex, as described above, which have a diameter of between 0.5 and ten microns, which can be aggregated into particles having a diameter of between 0.5 and ten microns.
  • the subject interferons are provided in liposomes or supramolecular complexes (such as described above) appropriately formulated for pulmonary delivery.
  • Polymers are preferably biodegradable within the time period over which release of the interferon is desired or relatively soon thereafter, generally in the range of one year, more typically a few months, even more typically a few days to a few weeks.
  • Biodegradation can refer to either a breakup of the microparticle, that is, dissociation of the polymers forming the microparticles and/or of the polymers themselves.
  • microparticles can be suspended in any appropriate pharmaceutical carrier, such as saline, for administration to a patient.
  • the microparticles will be stored in dry or lyophilized form until immediately before administration. They can then be suspended in sufficient solution, for example an aqueous solution for administration as an aerosol, or administered as a dry powder.
  • the microparticles can be delivered to specific cells, especially phagocytic cells and organs. Phagocytic cells within the Peyer's patches appear to selectively take up microparticles administered orally. Phagocytic cells of the reticuloendothelial system also take up microparticles when administered intravenously. Endocytosis of the microparticles by macrophages in the lungs can be used to target the
  • microparticles to the spleen, bone marrow, liver and lymph nodes are microparticles to the spleen, bone marrow, liver and lymph nodes.
  • microparticles can also be targeted by attachment of ligands, such as those described above, which specifically or non-specifically bind to particular targets.
  • ligands also include antibodies and fragments including the variable regions, lectins, and hormones or other organic molecules having receptors on the surfaces of the target cells.
  • the microparticles are stored lyophilized.
  • the dosage is determined by the amount of encapsulated interferon, the rate of release within the pulmonary system, and the pharmacokinetics of the compound.
  • microparticles can be delivered using a variety of methods, ranging from administration directly into the nasal passages so that some of the particles reach the pulmonary system, to the use of a powder instillation device, to the use of a catheter or tube reaching into the pulmonary tract.
  • Dry powder inhalers are commercially available, although those using hydrocarbon propellants are no longer used and those relying on the intake of a breath by a patient can result in a variable dose.
  • the subject invention provides a medical device having a coating adhered to at least one surface, wherein the coating comprises a polymer matrix and an interferon.
  • Such coatings can be applied to surgical implements such as screws, plates, washers, sutures, prosthesis anchors, tacks, staples, electrical leads, valves, membranes.
  • the devices can be catheters, implantable vascular access ports, blood storage bags, blood tubing, central venous catheters, arterial catheters, vascular grafts, intra-aortic balloon pumps, heart valves, cardiovascular sutures, artificial hearts, a pacemaker, ventricular assist pumps, extracorporeal devices, blood filters, hemodialysis units, hemoperfusion units, plasmapheresis units, and filters adapted for deployment in a blood vessel.
  • monomers for forming a polymer are combined with an interferon and are mixed to make a homogeneous dispersion of the interferon in the monomer solution.
  • the dispersion is then applied to a stent or other device according to a conventional coating process, after which the crosslinking process is initiated by a conventional initiator, such as UV light.
  • a polymer composition is combined with an interferon to form a dispersion.
  • the dispersion is then applied to a surface of a medical device and the polymer is cross-linked to form a solid coating.
  • a polymer and an interferon are combined with a suitable solvent to form a dispersion, which is then applied to a stent in a conventional fashion.
  • the solvent is then removed by a conventional process, such as heat evaporation, with the result that the polymer and interferon (together forming a sustained-release drug delivery system) remain on the stent as a coating.
  • a conventional process such as heat evaporation
  • An analogous process may be used where the interferon is dissolved in the polymer composition.
  • the device comprises an interferon and polymer suspension or dispersion, wherein the polymer is rigid, and forms a constituent part of a device to be inserted or implanted into a body.
  • the device is a surgical screw, stent, pacemaker, etc. coated with the interferon suspended or dispersed in the polymer.
  • the coating according to the present invention comprises a polymer that is bioerodible or non bioerodible.
  • the choice of bioerodible versus non-bioerodible polymer is made based upon the intended end use of the system or device.
  • the polymer is advantageously bioerodible.
  • the system is a coating on a surgically implantable device, such as a screw, stent, pacemaker, etc.
  • the polymer is advantageously bioerodible.
  • the polymer is advantageously bioerodible
  • the rate of bioerosion of the polymer is advantageously on the same order as the rate of interferon release.
  • the system comprises an interferon suspended or dispersed in a polymer that is coated onto a surgical implement, such as an orthopedic screw, a stent, a pacemaker, or a non-bioerodible suture
  • a surgical implement such as an orthopedic screw, a stent, a pacemaker, or a non-bioerodible suture
  • the polymer advantageously bioerodes at such a rate that the surface area of the interferon that is directly exposed to the surrounding body tissue remains substantially constant over time.
  • the polymer vehicle is permeable to water in the surrounding tissue, e.g. in blood plasma.
  • water solution may permeate the polymer, thereby contacting the interferon.
  • the rate of dissolution may be governed by a complex set of variables, such as the polymer's permeability, the solubility of the interferon, the pH, ionic strength, and protein composition, etc., of the physiologic fluid.
  • the polymer is non-bioerodible.
  • Non bioerodible polymers are especially useful where the system includes a polymer intended to be coated onto, or form a constituent part, of a surgical implement that is adapted to be permanently, or semi permanently, inserted or implanted into a body.
  • Exemplary devices in which the polymer advantageously forms a permanent coating on a surgical implement include an orthopedic screw, a stent, a prosthetic joint, an artificial valve, a permanent suture, a pacemaker, etc.
  • stents there is a multiplicity of different stents that may be utilized following percutaneous transluminal coronary angioplasty. Although any number of stents may be utilized in accordance with the present invention, for simplicity, a limited number of stents will be described in exemplary embodiments of the present invention. The skilled artisan will recognize that any number of stents may be utilized in connection with the present invention. In addition, as stated above, other medical devices may be utilized.
  • a stent is commonly used as a tubular structure left inside the lumen of a duct to relieve an obstruction.
  • stents are inserted into the lumen in a non- expanded form and are then expanded autonomously, or with the aid of a second device in situ.
  • a typical method of expansion occurs through the use of a catheter- mounted angioplasty balloon which is inflated within the stenosed vessel or body passageway in order to shear and disrupt the obstructions associated with the wall components of the vessel and to obtain an enlarged lumen.
  • a stent in accordance with the present invention may be embodied in a shape-memory material, including, for example, an appropriate alloy of nickel and titanium or stainless steel.
  • the interferon applied with enough specificity and a sufficient concentration to provide an effective dosage in the lesion area.
  • the "reservoir size" in the coating is preferably sized to adequately apply the interferon at the desired location and in the desired amount.
  • the entire inner and outer surface of the stent may be coated with the interferon in therapeutic dosage amounts. It is, however, important to note that the coating techniques may vary depending on the interferon. Also, the coating techniques may vary depending on the material comprising the stent or other intraluminal medical device.
  • the intraluminal medical device comprises the sustained release drug delivery coating.
  • the interferon coating may be applied to the stent via a conventional coating process, such as impregnating coating, spray coating and dip coating.
  • the interferon may be incorporated onto or affixed to the stent in a number of ways.
  • the interferon is directly incorporated into a polymeric matrix and sprayed onto the outer surface of the stent.
  • the interferon elutes from the polymeric matrix over time and enters the surrounding tissue.
  • the interferon preferably remains on the stent for at least three days up to approximately six months, and more preferably between seven and thirty days.
  • Example 1 Isolation of Human IFN ⁇ Clones [0188] Human interferon alpha sequences were isolated by PCR through standard methods.
  • the Human interferon alpha gene sequences identified using this approach are: G37 (SEQ ID NO: 1), G56 (SEQ ID NO: 3), G59 (SEQ ID NO: 5), G73 (SEQ ID NO: 7), G2O8 (SEQ ID NO: 9), G215 (SEQ ID NO: 1 1), G223 (SEQ ID NO: 13), G56M (SEQ ID NO: 15), G59M (SEQ ID NO: 17), and G215M (SEQ ID NO: 19).
  • PT37 (SEQ ID NO: 2), PT56 (SEQ ID NO: 4), PT59 (SEQ ID NO: 6), PT73 (SEQ ID NO: 8), PT208 (SEQ ID NO: 10), PT215 (SEQ ID NO: 12), PT223 (SEQ ID NO: 14), PT56M (SEQ ID NO: 16), PT59M (SEQ ID NO: 18), and PT215M (SEQ ID NO: 20).
  • the antiviral activity of the human IFN proteins was determined using the cytopathic effect (CPE) assay, as outlined in here.
  • CPE cytopathic effect
  • Human A549 epithelial carcinoma cells ATCC Manassas, VA
  • ATCC Manassas, VA were plated in a 96 well plate at 20,000 cells/well and grown in 0.1 ml DMEM with 10% Fetal Bovine Serum. Two to four hours later 0.1 ml of serial dilutions of IFN samples were added and the plate was incubated overnight. The cells were then infected with 0.05 ml of
  • encephalomyocarditis virus and returned to the incubator for 40-48 hours. Once killing was complete in wells which contained no interferon the media was removed and the remaining cells were stained with crystal violet and washed gently with tapwater to remove excess dye. The dye was then released by addition of 0.1 ml of 70% methanol or ethanol and read at 570 or 562 nm on a Molecular Devices plate reader. The percent cell survival was determined by subtracting the virus control (no IFN) from each sample and taking 100% protection as the cell control (no IFN, no virus). The EC50 was determined using GraphPad Prism software using a Sigmoidal, variable slope curve fit.
  • NK-92 The natural killer phenotype Human non-Hodgkin's lymphoma cell line NK-92 (ATCC Manassas, VA) was maintained in the ATCC-recommended media containing 100-200 U/ml 1L-2. Prior to assay with IFN protein, the cells were washed 2X with media without IL-2 and then 50,000 cells in 0.1 were added to a microtiter plate. Serial dilution of IFN samples were prepared and 0.15 ml was added to the cells. After incubation for 24 hours the plate was centrifuged to pellet the cells and the supernatant removed and frozen for later analysis.
  • IFN-gamma production was determined by a commercial ELISA (BL InterferonSource# 41500) and the data analyzed in the GraphPad Prism software package. The curve fitting was set to place the bottom of the curve at the background of IFN-gamma produced by cells which had not been stimulated by IFN, and the top of the curve set as the maximal IFN-gamma produced in the experiment. All assays were run concurrently with a defined human IFN alpha-2a standard. The NK-92 cell serves as a model for stimulation of immune cell activity. 101941 Example 4: Antiproliferative Activity of the IFN Proteins and Mutants on Human Fetal Lung Fibroblast Cell Line HFLl
  • the human fetal lung fibroblast cell line HFLl (ATCC Manassas, VA) was maintained in F12K media with 10% FBS and was plated in a microtiter plate at 2500 cells in 0.1 ml. The plate was then incubated at 37 degrees C to allow the cells to adhere. The media was then removed and 0.2 ml of serially diluted IFN sample was added to the cells. The plate was then returned to the incubator and the cells were allowed to grow for 6 to 7 days. At this point 0.1 ml of the media was removed and 0.06 ml of MTS reagent (Promega) was added to each well and allowed to incubate for 4 hours.
  • MTS reagent Promega
  • the plate was then read at 490 nm in a Molecular Devices plate reader.
  • the data was analyzed in the GraphPad Prism software package taking 100% as the value obtained for cells grown without IFN and 0% as the background of MTS added to media with no cells. All assays were run concurrently with a defined IFN alpha-2a standard.
  • the HFLl cell serves as a model for antiproliferative activity against putatively normal human cells.
  • the human ovarian carcinoma cell line NIH:OCVAR-3 (ATCC Manassas, VA) was maintained in the ATCC recommended media.
  • ATCC Manassas, VA ATCC Manassas, VA
  • cells in 0.1 ml were plated in a 96 well plate which was incubated at 37 degrees C for 4 hours to allow the cells to adhere. After 4 hours the media was removed and 0.2 ml of serial dilutions of IFN samples were added to the cells. These were returned to the incubator and allowed to grow for 6 to 7 days. At this point 0.1 ml of media was removed and 0.06ml of MTS reagent (Promega) was added to the media and allowed to incubate for 4 hours.
  • MTS reagent Promega
  • Clinical benefits may include, but are not limited to, an enhanced therapeutic window, i.e., the difference between the dose required to have a beneficial clinical effect and the dose at which dose-limiting side effects become evident, apparent with these molecules as assessed in vitro.
  • an enhancement in the therapeutic window may be more evident in certain patient subpopulations and may relate to patients' common or unique pharmacogenomic profiles or prior exposure either to pharmacological agents or to environmental factors.
  • IFNs may be useful as IFN antagonists is certain conditions where reduction or modulation of endogenous IFN activity is warranted.

Abstract

L'invention porte sur un nouvel ensemble de polypeptides et de gènes d'interférons. Les nouveaux polypeptides d'interférons sont actifs et seront utiles à des fins thérapeutiques. L'administration des gènes d'interférons peut également être utile à des fins thérapeutiques.
PCT/US2010/043536 2009-07-28 2010-07-28 Protéines et gènes d'interférons humains mutant WO2011017160A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US22916709P 2009-07-28 2009-07-28
US61/229,167 2009-07-28

Publications (1)

Publication Number Publication Date
WO2011017160A1 true WO2011017160A1 (fr) 2011-02-10

Family

ID=43544600

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/043536 WO2011017160A1 (fr) 2009-07-28 2010-07-28 Protéines et gènes d'interférons humains mutant

Country Status (1)

Country Link
WO (1) WO2011017160A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108912222A (zh) * 2018-08-02 2018-11-30 中国农业科学院北京畜牧兽医研究所 一种重组犬干扰素CaIFN-λ及其应用
WO2022079205A1 (fr) * 2020-10-15 2022-04-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation de polypeptides d'ifn-alpha pour le traitement d'infections à coronavirus
CN114891087A (zh) * 2022-04-26 2022-08-12 浙江皇冠科技有限公司 草鱼干扰素、草鱼干扰素突变体及其应用和产品
US11440943B2 (en) * 2019-03-28 2022-09-13 Orionis Biosciences, Inc. Therapeutic interferon alpha 1 proteins
EP3349783B1 (fr) * 2015-09-15 2024-01-17 ILC Therapeutics Ltd Compositions et méthodes associées au traitement de maladies

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040105841A1 (en) * 2000-11-03 2004-06-03 Sidney Pestka Interferons, uses and compositions related thereto
US20060094055A1 (en) * 2002-05-15 2006-05-04 The Regents Of The University Of California RNA silencing in animals as an antiviral defense
US7358333B2 (en) * 2001-05-03 2008-04-15 Genodysse S.A. Polypeptides of the IFNα-5 gene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040105841A1 (en) * 2000-11-03 2004-06-03 Sidney Pestka Interferons, uses and compositions related thereto
US7358333B2 (en) * 2001-05-03 2008-04-15 Genodysse S.A. Polypeptides of the IFNα-5 gene
US20060094055A1 (en) * 2002-05-15 2006-05-04 The Regents Of The University Of California RNA silencing in animals as an antiviral defense

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3349783B1 (fr) * 2015-09-15 2024-01-17 ILC Therapeutics Ltd Compositions et méthodes associées au traitement de maladies
CN108912222A (zh) * 2018-08-02 2018-11-30 中国农业科学院北京畜牧兽医研究所 一种重组犬干扰素CaIFN-λ及其应用
CN108912222B (zh) * 2018-08-02 2020-06-19 中国农业科学院北京畜牧兽医研究所 一种重组犬干扰素CaIFN-λ及其应用
US11440943B2 (en) * 2019-03-28 2022-09-13 Orionis Biosciences, Inc. Therapeutic interferon alpha 1 proteins
WO2022079205A1 (fr) * 2020-10-15 2022-04-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Utilisation de polypeptides d'ifn-alpha pour le traitement d'infections à coronavirus
CN114891087A (zh) * 2022-04-26 2022-08-12 浙江皇冠科技有限公司 草鱼干扰素、草鱼干扰素突变体及其应用和产品
CN114891087B (zh) * 2022-04-26 2023-08-11 浙江皇冠科技有限公司 草鱼干扰素、草鱼干扰素突变体及其应用和产品

Similar Documents

Publication Publication Date Title
AU2004270102B2 (en) Uses of interferons for the treatment of severe acute respiratory syndrome and other viral infections
JP5914356B2 (ja) インターフェロンアナログ
CN108473555A (zh) 条件活性多肽
JP2014508515A (ja) Ｃｘｃｒ４細胞に治療分子を効率的かつ選択的に送達するための方法および試薬
CN110121506A (zh) 条件活性多肽及产生它们的方法
WO2011017160A1 (fr) Protéines et gènes d'interférons humains mutant
TWI694084B (zh) Ｕｔｉ融合蛋白質
WO2020154640A1 (fr) Compositions et méthodes de stimulation de cellules tueuses naturelles
TW200900504A (en) Recombinant human interferon-like proteins
US20100267139A1 (en) Osteopontin nanoparticle system for drug delivery
IL155741A (en) Interferons,their uses and compositions related thereto
JP2001526689A (ja) トロンボポエチンの新規な投与
US8501680B2 (en) Antagonists against interaction of PF4 and RANTES
US11571455B2 (en) Methods and compositions for treating alcoholic liver disease
KR102274876B1 (ko) 신규한 세포 투과성 펩타이드 및 이의 용도
US6562334B1 (en) Canine interleukin 12 administration as an immune disease treatment
EP2229179B1 (fr) Récepteur ldl soluble dans l'hépatite virale
KR102274877B1 (ko) 신규한 세포 투과성 펩타이드 및 이의 용도
CN117209612A (zh) 融合蛋白及其应用
Liu et al. Surface engineering of liposomal formulations for targeted drug delivery
WO2021243148A2 (fr) Méthodes et compositions de traitement de la maladie alcoolique du foie
EP3923975A2 (fr) Ciblage d'une sous-population tnfr2 + cdc2 (r2d2) résidant dans les poumons pour traiter l'asthme
KR20230019109A (ko) Akt3 조절제 및 이의 사용 방법
DE10240894A1 (de) Verstärkung der Resorption von Subtanzen über die Haut und Schleimhaut
SUZUKI et al. Immunomodulating Antitumor

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10806924

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10806924

Country of ref document: EP

Kind code of ref document: A1