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
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  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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  • A61P25/00—Drugs for disorders of the nervous system
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  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
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  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
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• • A—HUMAN NECESSITIES
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  • A61P31/12—Antivirals
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• • A—HUMAN NECESSITIES
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  • A61P31/12—Antivirals
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to the generation of new interferon -alpha homologues.
• Interferon-alphas are members of the diverse helical-bundle superfamily of cytokine genes (Sprang, S.R. et al. (1993) Curr. Opin. Struct. Biol. 3:815-827).
• the human interferon-alphas are encoded by a family of over 20 tandemly duplicated nonallelic genes that share 85-98% sequence identity at the amino acid level (Henco, K. et al. (1985) J Mol. Biol. 185:227-260).
• Interferon-alphas have been shown to inhibit various types of cellular proliferation, and are especially useful for the treatment of a variety of cellular proliferation disorders frequently associated with cancer, particularly hematologic malignancies such as leukemias.
• These proteins have shown antiproliferative activity against multiple myeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronic myelogenous leukemia, renal-cell carcinoma, urinary bladder tumors and ovarian cancers (Bonnem, E.M. et al. (1984) J. Biol. Response Modifiers 3:580; Oldham, R.K. (1985) Hospital Practice 20:71).
• Interferon-alphas are also useful against various types of viral infections (Finter, N.B. et al. (1991) Drugs 42(5):749). Interferon-alphas have shown activity against human papillomavirus infection, Hepatitis B, and Hepatitis C infections (Finter, N.B. et al., 1991, supra; Kashima, H. et al. (1988) Laryngoscope 98:334; Dusheiko, G.M. et al. (1986) J. Hematology 3 (Supple. 2):S199; Davis, GL et al. (1989) N. England J. Med. 321:1501). The role of interferons and interferon receptors in the pathogenesis of certain autoimmune and inflammatory diseases has also been investigated (Benoit, P. et al. (1993) J. Immunol. 150(3):707).
• the invention provides novel interferon-alpha (IFN-alpha or IFN- ⁇ ) homologue polypeptides, nucleic acids encoding the polypeptides and complementary nucleotide sequences thereof, fragments of said polypeptides and nucleic acids, antibodies to the polypeptides, and uses therefor, data sets containing character strings of interferon- alpha homologue sequences, and automated systems for using the character strings.
• the invention includes an isolated or recombinant interferon- alpha nucleic acid homologue.
• polynucleotide sequences selected from SEQ ID NO:l to SEQ ID NO:35, or to SEQ ID NO:72 to SEQ ID NO:78, and complementary polynucleotide sequences thereof.
• Polynucleotide sequences encoding a polypeptide selected from SEQ ID NO:36 to SEQ ID NO: 81 or from SEQ ID NO:79 to SEQ ID NO: 85, and complementary polynucleotide sequences thereof are also a feature of the invention.
• a polynucleotide sequence which hybridizes under highly stringent conditions over substantially the entire length of any of the preceding polynucleotide sequences is a feature of the present invention.
• a polynucleotide sequence comprising a nucleotide fragment of any of the preceding polynucleotide sequences which nucleotide fragment encodes a polypeptide having an antiproliferative activity in a human Daudi cell line- based cell proliferation assay is a feature of the invention.
• a polynucleotide sequence comprising a nucleotide fragment of any of the polynucleotide sequences of the invention described above and below which encodes a polypeptide having antiviral activity in a murine cell line/EMCV - based assay is a feature of the invention.
• the invention also includes an isolated or recombinant nucleic acid, comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises the amino acid sequence: CDLPQTHSLG-X ⁇ -X 12 -RA-X 15 -X 1 -LL-X 19 -QM- X2 2 -R-X24-S-X 2 6-FSCLKDR-X3 4 -DFG-XSS-P-X 4 0-EEFD-X 4 5-X 4 0-X 47 -FQ-X50-X S 1 -QAI- X 55 -X 56 -X 57 -HE-X 60 -X 6 ⁇ -QQTFN-X 67 -FSTK-X 72 -SS-X 75 -X 76 -W-X 78 -X 79 -X 80 -LL-X 83 -K- Xgs-Xse-T-Xss-L-Xw-QQLN-Xg
• WEVVRAEIMRSFSFSTNLQKRLRRKE or a conservatively substituted variation thereof, where X] i is N or D; X 12 is R, S, or K; X 15 is L or M; X 16 is I, M, or V; X ⁇ 9 is A or G; X 22 is G or R; X 2 is I or T; X 26 is P or H; X 34 is H, Y or Q; X 38 is F or L; 40 is Q or R; X 15 is G or S; 6 is N or H; X 47 is Q or R; X 50 is K or R; X 1 is A or T; X 55 is S or F; X 56 is V or A; X 57 is L or F; X 6 o is M or I; X 61 is I or M; X 67 is L or F; X 72 is D or N; X 75 is A or V; X 7 is A or T; X 78 is E or D; X 7 is Q or
• each of the single letters of this amino acid sequence represents a particular amino acid residue according to standard practice known to those of ordinary skill in the art.
• the encoded polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:36 to SEQ ID NO:54; and the nucleic acid comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:l to SEQ ID NO: 19.
• the invention also provides polypeptide fragments of any of SEQ NOS:36- 70 and SEQ ID NOS:72-79.
• such a polypeptide fragment exhibits an antiproliferative activity in a human Daudi cell line- based cell proliferation assay or an antiviral activity in a murine cell line/EMCV - based assay, or both said activities.
• the human Daudi cell line- based cell proliferation assay and antiviral activity in a murine cell line/EMCV - based assay are described in greater detail below.
• the invention provides a polynucleotide sequence comprising a nucleotide fragment of any nucleic acid of the invention described above and below, wherein said nucleotide fragment encodes a polypeptide fragment that exhibits an antiproliferative activity in a human Daudi cell line- based cell proliferation assay or an antiviral activity in a murine cell line/EMCV - based assay, or both activities, as is described in greater detail below.
• the invention also includes an isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence comprising at least 20 contiguous amino acids of any one of SEQ ID NOS:36-70.
• the polypeptide of the invention comprises an amino acid sequence comprising one or more of amino acid residues (Tyr or Gln)34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ilel32, Argl34, Phel52, Lysl ⁇ O, and Glul66, wherein the numbering of the amino acid residues corresponds to the numbering of residues in the amino acid sequence of SEQ ED NO:36.
• the encoded polypeptide of the invention comprises at least 30, at least 50, at least 70, at least 75, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 155, at least 160, or at least 165 contiguous amino acid residues of any one of SEQ ID NOS:36-70.
• the encoded polypeptide is at least 150, at least 155, at least 160, at least 163, or at least 165 amino acids in length.
• the encoded polypeptide is about 166 amino acids in length.
• the encoded polypeptide comprises an amino acid sequence selected from SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ED NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:45, and SEQ ID NO:46.
• the invention provides a nucleic acid that comprises a polynucleotide sequence selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ED NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 10, and SEQ ID NO:l l.
• polypeptide encoded by any nucleic acid or the invention described herein or a fragment thereof may have antiproliferative activity in a human Daudi cell line - based assay, or antiviral activity in a human WISH cell EMCV- based assay.
• the encoded polypeptide has antiproliferative activity of at least about 8.3xl0 6 units/milligram in the human Daudi cell line - based assay (1 unit is the amount of protein in milligram (mg) required to induce 50% antiproliferative activity), or antiviral activity of at about least 2.1xl0 7 units/milligram (mg) in the human WISH cell/EMCV-based assay (1 unit is the amount of protein in mg required to induce 50% antiviral activity).
• the encoded polypeptide can bind to a type I interferon receptor, preferably a human type I interferon receptor, more preferably a human (e.g., type I) interferon-alpha receptor.
• the invention also includes a cell comprising any nucleic acid of the invention described herein, or which expresses any polypeptide of the invention noted herein.
• the cell expresses a polypeptide encoded by the nucleic acid of the invention as described herein.
• the invention also includes a vector comprising any nucleic acid of the invention described above and below.
• the vector can comprise a plasmid, a cosmid, a phage, or a virus; the vector can be, e.g., an expression vector, a cloning vector, a packaging vector, an integration vector, or the like.
• the invention also includes a cell transduced by a vector of the invention.
• the invention also includes compositions comprising any nucleic acid of the invention described above and below, and an excipient, preferably a pharmaceutically acceptable excipient.
• Cells and transgenic animals which include any polypeptide or nucleic acid of the invention described above and below, e.g., produced by transduction of vector, are a feature of the invention.
• the invention also includes compositions produced by digesting one or more of the nucleic acids of the invention described above or below with a restriction endonuclease, an RNAse, or a DNAse; and, compositions produced by incubating one or more nucleic acids described above or below in the presence of deoxyribonucelotide triphosphates and a nucleic acid polymerase, e.g., a thermostable polymerase.
• a nucleic acid polymerase e.g., a thermostable polymerase.
• the invention also includes compositions comprising two or more nucleic acids described above or below.
• the composition may comprise a library of nucleic acids, where the library contains at least about 5, 10, 20 or 50 nucleic acids.
• the invention includes an isolated or recombinant polypeptide encoded by any nucleic acid described above or below.
• the polypeptide may comprise a sequence selected from SEQ ID NO:36 to SEQ ID NO:70, or SEQ ID NO:79 to SEQ ID NO:85.
• the invention also includes a polypeptide comprising at least 50 contiguous amino acids of a protein encoded by a polynucleotide sequence, the polynucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 1 to SEQ ID NO:35 or SEQ ID NO:72 to SEQ ID NO:78; (b) a polynucleotide sequence that encodes a polypeptide selected from SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85; and (c) a complementary sequence of a polynucleotide sequence which hybridizes under highly stringent conditions over substantially the entire length of polynucleotide sequence (a) or (b).
• the polypeptide comprises at least about 70, 100, 120, 130, 140, 150, 155, 160, 165, or 166 contiguous amino acids of the encoded protein.
• the invention also includes an isolated or recombinant polypeptide comprising an amino acid sequence comprising at least 50 contiguous amino acid residues of any one of SEQ ID NOS:36-70, and one or more of amino acids Alal9, (Tyr or Gln)34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ilel32, Argl34, Phel52, Lysl ⁇ O, and Glul66, where the numbering of the amino acids corresponds to that of SEQ ID NO:36.
• the polypeptide comprises at least about 50, 70, 75, 100, 110, 120, 130, 140 150, 155, 160, 163, 165, or 166 contiguous amino acids of any one of SEQ ID NOS:36-70. In more preferred embodiments, the polypeptide comprises at least about 50, 70, 75, 100, 110, 120, 130, 140, 150, 155, 160, 163, 165, or 166 contiguous amino acid residues of any one of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:45, or SEQ ID NO:46.
• the polypeptide of the invention is at least about 50, 70, 75, 100, 110, 120, 130, 140, 150, 155, 160, 163, 165, or 166 amino acid residues in length, or is preferably 166 amino acids in length.
• Longer polypeptides e.g., which comprise purification tags or the like, are also contemplated. Such polypeptides may display antiproliferative activities in human Daudi cell-line based assay and/or antiviral activities in a human WISH cell/EMCV-based assay.
• the invention also includes a polypeptide which specifically binds polyclonal antisera raised against at least one antigen, said at least one antigen comprising a polypeptide sequence selected from an amino acid sequence set forth in SEQ ID NO: 36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85 or a fragment thereof.
• the invention provides polypeptides which bind a polyclonal antisera raised against at least one antigen, wherein said at least one antigen comprises at least one amino acid sequence set forth in SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85, or a fragment of any of these amino sequences, wherein the polyclonal antisera is subtracted with one or more known interferon-alpha polypeptides or proteins, including, e.g., a polypeptide or protein encoded by a nucleic acid having or corresponding to one or more of the following GenBankTM accession numbers: J00210 (alpha-D), J00207 (Alpha-a), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha- 14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21
• Any polypeptide described above or below optionally has antiproliferative activity in a human Daudi cell line - based assay and/or in an antiviral activity in a human WISH cell/EMCV-based assay. Any polypeptide described above or below can have antiproliferative activity of at least about 8.3xl0 6 units/mg in the human Daudi cell line -
• any polypeptide described above or below can bind to a type I interferon receptor, preferably a human type I interferon receptor, more preferably a human interferon-alpha receptor.
• any polypeptide described above or below may further include a secretion/localization sequence, e.g., a signal sequence, an organelle targeting sequence, a membrane localization sequence, and the like.
• Any polypeptide described herein may further include a sequence that facilitates purification, e.g., an epitope tag (such as, a FLAG epitope), a polyhistidine tag, a GST fusion, and the like.
• the polypeptide optionally includes a methionine at the N-terminus.
• Any polypeptide of the invention described herein optionally includes one or more modified amino acids, such as a glycosylated amino acid, a PEG-ylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, an acylated amino acid, or the like.
• compositions comprising any polypeptide described herein in an excipient, preferably a pharmaceutically acceptable excipient.
• the invention also includes an antibody or antisera produced by administering one or more of the polypeptides of the invention described herein to a mammal, wherein the antibody or antisera does not specifically bind to a known alpha- interferon polypeptide or protein, including, e.g., any polypeptide or protein encoded by a nucleic acid having or corresponding to one or more of the following GenBank accession numbers: J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (TFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), R0067 (Gx-1), 101614,
• the invention also includes antibodies which specifically bind a polypeptide comprising a sequence selected from SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85.
• the antibodies are, e.g., polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments, fragments produced by an Fab expression library, or the like.
• Methods for producing the polypeptides of the invention are also included.
• One such method comprises introducing into a population of cells any nucleic acid described herein, operatively linked to a regulatory sequence effective to produce the encoded polypeptide, culturing the cells in a culture medium to produce the polypeptide, and optionally isolating the polypeptide from the cells or from the culture medium.
• the nucleic acid may be part of a vector, such as a recombinant expression vector.
• the invention also includes a method of inhibiting growth of tumor cells, by contacting the tumor cells with a polypeptide of the invention described herein, thereby inhibiting growth of the tumor cells.
• the invention includes a method of inhibiting growth of population of tumor cells comprising contacting the population of tumor cells with an effective amount of a polypeptide of the invention sufficient to inhibit growth of tumor cells in said population of tumor cells, thereby inhibiting growth of tumor cells in said population of cells.
• the tumor cells can be human carcinoma cells, human leukemia cells, human T-lymphoma cells, human melanoma cells, other human cancer cells as described herein, and the like.
• the tumor cells can be in vivo, ex vivo, or in vitro (e.g., cultured cells).
• the invention also includes a method of inhibiting the replication of a virus within one or more cells infected by the virus, by contacting one or more of the infected cells with an effective amount of a polypeptide of the invention as described above and below, wherein said amount is sufficient to inhibit viral replication in said one or more infected cells, thereby inhibiting replication of the virus in the one or more cells.
• the virus can be an RNA virus, e.g., a human immunodeficiency virus or a hepatitis C virus, or a DNA virus, e.g., a hepatitis B virus.
• the infected cells can be in vivo, ex vivo, or in vitro (e.g., cultured cells).
• the invention also includes a method of treating an autoimmune disorder in a subject in need of such treatment, by administering to the subject an effective amount of a polypeptide of the invention as described herein sufficient to treat the autoimmune disorder.
• the autoimmune disorder may be multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type I diabetes, and the like.
• the invention also includes, in a method of treating a disorder treatable by administration of interferon- alpha to a subject, an improvement comprising administering to the subject an effective amount of a polypeptide of the invention as described herein sufficient to treat said disorder.
• the disorder treatable by administration of interferon-alpha disorder may be multiple sclerosis, rheumatoid arthritis, lupus erythematosus, type I diabetes, AIDS or AEDS-related complexes, or the like.
• nucleic acids and proteins derived by mutation of the sequences herein are a feature of the invention.
• those produced by diversity generation or recursive sequence recombination (RSR) methods e.g., DNA shuffling
• RSR recursive sequence recombination
• Mutation and recombination methods using the nucleic acids described herein are a feature of the invention.
• one method of the invention includes recursively recombining one or more nucleic acid sequences of the invention as described above and below with one or more additional nucleic acids (including, but not limited to, those noted herein), each sequence of the one or more additional nucleic acids encoding an interferon-alpha homologue or an amino acid subsequence thereof.
• the recombining steps are optionally performed in vivo, ex vivo, in silico or in vitro. Said recursive recombination produces at least one library of recombinant interferon-alpha homologue nucleic acids.
• a recombinant interferon-alpha homologue nucleic acid produced by this method a cell containing the recombinant interferon-alpha homologue nucleic acid, a nucleic acid library produced by this recursive recombination method, a composition comprising two or more of said recombinant interferon-alpha nucleic acids, and a population of cells comprising such recombinant interferon-alpha nucleic acids or containing the library.
• the library comprise at least ten such recombinant nucleic acids.
• the invention also provides a method of producing a modified or recombinant interferon-alpha homologue nucleic acid that comprises mutating a nucleic acid of the invention as described herein.
• nucleic acids that encode an interferon-alpha homologue having an increased growth inhibition activity, cytostatic activity, or cytotoxic activity against a population of cells (e.g., cancer cells) relative to the growth inhibition activity cytostatic activity, or cytotoxic activity, respectively, of human interferon-alpha 2a or other known interferon-alpha against the population of cells.
• Figures 1A-1E show an alignment of exemplary mature interferon homologue polypeptide sequences (SEQ ID NOS: 36-70 and 79-85) according to the invention.
• Figure 2 shows antiproliferative activities in a human Daudi cell line - based assay and antiviral activities in a human WISH cell/EMCV-based assay of, respectively, exemplary interferon homologues of the present invention relative to the respective antiproliferative and antiviral activities of two control compounds, human interferon alpha-2a ("IFN- ⁇ -2a" or "2a") and consensus human interferon ("IFN-Conl” or "Conl”).
• IFN- ⁇ -2a human interferon alpha-2a
• 2a consensus human interferon
• FIG. 3A, 3B, and 3C illustrate activity profiles of EFN-alpha homologue 3DA11 (SEQ ID NO:40) and control interferons, human interferon al ⁇ ha-2a ( “2a”) and consensus human interferon alpha ( "Conl”), against a panel of tumor cell lines.
• SEQ ID NO:40 EFN-alpha homologue 3DA11
• 2a human interferon al ⁇ ha-2a
• Consl consensus human interferon alpha
• FIG. 3A shows the cell total growth inhibitory activity of IFN-alpha homologue 3DA11 and each control IFN on each respective cell line as reflected in the GI50 value, which is the concentration ( ⁇ g/ml) of interferon alpha homologue or control IFN alpha at which growth of a particular cell line is inhibited by 50%, as measured by a 50% reduction in the net pro tein/polypep tide increase in the interferon alpha homologue or control EFN alpha at the end of the incubation period.
• Fig. 3B shows the cytostatic activity of IFN-alpha homologue 3DA11 and each control EFN on each cell line of the panel of cell lines. Cytostatic activity refers to an activity capable of suppressing growth and multiplication of cells.
• Cytostatic activity is assessed as a reflection of the concentration of IFN-alpha homologue 3DA11 or control IFN ( ⁇ g/ml) at which the growth and/or multiplication of cells of a particular cell line is completely inhibited or suppressed, such that the amount of cellular protein at the end of the incubation period equals the amount of cellular protein at the beginning of the incubation period ("total growth inhibition" or "TGI").
• Fig. 3C illustrates the cytotoxic activity of IFN-alpha homologue 3DA11 and each control IFN on each respective cell line.
• the cytotoxicity of an agent is the degree to which the agent possess a specific destructive action on certain cells or the possession of such action.
• the term typically refers to an agent capable of causing cell death and is used particularly in referring to the lysis of cells by immune phenomena and to agents of compounds that selectively kill dividing cells.
• cytotoxic activity is illustrated as LC50, the concentration of IFN-alpha homologue 3DA11 ( ⁇ g/ml) at which a 50% reduction in the net protein increase in control cells (control IFN alpha) at the end of the incubation as compared to that at the beginning of the incubation period is observed, indicating a net loss of cells following addition of the particular interferon. Cytotoxic activity may be assessed as the concentration of IFN-alpha homologue 3DA11 at which, relative to the control cells, 50% of the total number of cells (i.e., total population) of a particular cell line are destroyed or killed.
• Figs. 4A, 4B, 4C, and 4D show the cytostatic activity of selected interferon-alpha homologues of the present invention relative to the cytostatic activities of two control interferon alphas, human interferon-alpha 2a ("2a") and consensus human interferon-alpha ("Conl”), against a leukemia cell line (RPMI-8226) (Fig. 4A), a lung cancer cell line (NCI-H23) (Fig. 4B), a renal cancer cell line (ACHN) (Fig. 4C), and an ovarian cancer cell line (OVCAR-3) (Fig. 4D), respectively.
• RPMI-8226 a leukemia cell line
• NCI-H23 a lung cancer cell line
• ACBN renal cancer cell line
• OFC ovarian cancer cell line
• OFVCAR-3 ovarian cancer cell line
• Cytostatic activity is reflected by a TGI value for a particular interferon alpha (i.e., the concentration of interferon alpha at which cell growth of a cell line is totally inhibited, wherein the amount of cellular protein at the end of the incubation period equals the amount of cellular protein at the beginning of the incubation period).
• Fig. 5 presents a comparison of the number of mice (out of a total number of six mice) that survived following administration of doses of 2 ⁇ g, 10 ⁇ g, and 50 ⁇ g of two exemplary IFN-alpha homologues of the present invention (designated "IFN-CH2.2" and “IFN-CH2.3”), doses of 2 ⁇ g, 10 ⁇ g, and 50 ⁇ g of murine IFN-alpha-4, and doses of 2 ⁇ g, 10 ⁇ g, and 50 ⁇ g of human IFN-alpha-2a, respectively.
• the results shown in Fig. 5 demonstrate that in a murine model system, the improved in vitro antiviral activity of these two exemplary IFN-alpha homologues is maintained and sustained in vivo.
• PBS Phosphate- buffered saline
• a "polynucleotide sequence” is a nucleic acid (which is a polymer of nucleotides (A,C,T,U,G, etc. or naturally occurring or artificial nucleotide analogues)) or a character string representing a nucleic acid, depending on context. Either the given nucleic acid or the complementary nucleic acid can be determined from any specified polynucleotide sequence.
• amino acid sequence is a polymer of amino acids (a protein, polypeptide, etc.) or a character string representing an amino acid polymer, depending on context. Either the given nucleic acid or the complementary nucleic acid can be determined from any specified polynucleotide sequence.
• a nucleic acid, protein, peptide, polypeptide, or other component is
• nucleic acid, polypeptide, or other component is isolated when it is partially or completely recovered or separated from other components of its natural environment such that it is the predominant species present in a composition, mixture, or collection of components (i.e., on a molar basis it is more abundant than any other individual species in the composition).
• the preparation consists of more than 70%, typically more than 80%, or preferably more than 90% of the isolated species.
• a "substantially pure” or “isolated” nucleic acid e.g., RNA or DNA
• polypeptide, protein, or composition also means where the object species (e.g., nucleic acid or polypeptide) comprises at least about 50, 60, or 70 percent by weight (on a molar basis) of all macromolecular species present.
• a substantially pure or isolated composition can also comprise at least about 80, 90, or 95 percent by weight of all macromolecular species present in the composition.
• An isolated object species can also be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of derivatives of a single macromolecular species.
• isolated nucleic acid may refer to a nucleic acid (e.g., DNA or RNA) that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5' and one at the 3' end) in the naturally occurring genome of the organism from which the nucleic acid of the invention is derived.
• a nucleic acid e.g., DNA or RNA
• this term includes, e.g., a cDNA or a genomic DNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease treatment, whether such cDNA or genomic DNA fragment is incorporated into a vector, integrated into the genome of the same or a different species than the organism, including, e.g., a virus, from which it was originally derived, linked to an additional coding sequence to form a hybrid gene encoding a chimeric polypeptide, or independent of any other DNA sequences.
• the DNA may be double-stranded or single-stranded, sense or antisense.
• a nucleic acid or polypeptide is "recombinant" when it is artificial or engineered, or derived from an artificial or engineered protein or nucleic acid.
• the term “recombinant” when used with reference e.g., to a cell, nucleotide, vector, or polypeptide typically indicates that the cell, nucleotide, or vector has been modified by the introduction of a heterologous (or foreign) nucleic acid or the alteration of a native nucleic acid, or that the polypeptide has been modified by the introduction of a heterologous amino acid, or that the cell is derived from a cell so modified.
• Recombinant cells express nucleic acid sequences (e.g., genes) that are not found in the native (non-recombinant) form of the cell or express native nucleic acid sequences (e.g., genes) that would be abnormally expressed under-expressed, or not expressed at all.
• the term "recombinant nucleic acid” e.g., DNA or RNA) molecule means, for example, a nucleotide sequence that is not naturally occurring or is made by the combatant (for example, artificial combination) of at least two segments of sequence that are not typically included together, not typically associated with one another, or are otherwise typically separated from one another.
• a recombinant nucleic acid can comprise a nucleic acid molecule formed by the joining together or combination of nucleic acid segments from different sources and/or artificially synthesized.
• the term "recombinantly produced” refers to an artificial combination usually accomplished by either chemical synthesis means, recursive sequence recombination of nucleic acid segments or other diversity generation methods (such as, e.g., shuffling) of nucleotides, or manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques known to those of ordinary skill in the art.
• Recombinantly expressed typically refers to techniques for the production of a recombinant nucleic acid in vitro and transfer of the recombinant nucleic acid into cells in vivo, in vitro, or ex vivo where it may be expressed or propagated.
• a "recombinant polypeptide” or “recombinant protein” usually refers to polypeptide or protein, respectively, that results from a cloned or recombinant gene or nucleic acid.
• a "subsequence” or “fragment” is any portion of an entire sequence, up to and including the complete sequence.
• Numbering of a given amino acid or nucleotide polymer “corresponds to numbering" of a selected amino acid polymer or nucleic acid when the position of any given polymer component (amino acid residue, inco ⁇ orated nucleotide, etc.) is designated by reference to the same residue position in the selected amino acid or nucleotide, rather than by the actual position of the component in the given polymer.
• a vector is a composition for facilitating cell transduction by a selected nucleic acid, or expression of the nucleic acid in the cell.
• Vectors include, e.g., plasmids, cosmids, viruses, YACs, bacteria, poly-lysine, etc.
• An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
• the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
• the expression vector typically includes a nucleic acid to be transcribed operably linked to a promoter.
• substantially an entire length of a polynucleotide or amino acid sequence refers to at least about 50%, at least about 60%, generally at least about 70%, generally at least about 80%, or typically at least about 90%, 95,%, 96%, 91%, 98%, or 99% or more of a length of an amino acid sequence or nucleic acid sequence.
• a human alpha-interferon receptor is a receptor which is naturally activated in human cells by an alpha interferon.
• Naturally occurring refers to the fact that the object can be found in nature.
• a polypeptide or polynucleotide sequence that is present in an organism, including viruses, that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
• a "naturally occurring" nucleic acid (e.g., DNA or RNA) molecule is a nucleic acid molecule that exists in the same state as it exists in nature; that is, the nucleic acid molecule is not isolated, recombinant, or cloned.
• an “antibody” refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.
• Light chains are classified as either kappa or lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• a typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer.
• Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
• the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• the terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.
• Antibodies exist as intact immunoglobuhns or as a number of well characterized fragments produced by digestion with various peptidases.
• pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond.
• the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')2 dimer into an Fab' monomer.
• the Fab' monomer is essentially an Fab with part of the hinge region (see Fundamental Immunology, W.E. Paul, ed., Raven Press, N.Y. (1993), for a more detailed description of other antibody fragments).
• antibody includes single chain antibodies, including single chain Fv (sFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide.
• sFv single chain Fv
• An "antigen-binding fragment" of an antibody is a peptide or polypeptide fragment of the antibody which binds an antigen.
• An antigen-binding site is formed by those amino acids of the antibody which contribute to, are involved in, or affect the binding of the antigen. See Scott, T.A. and Mercer, E.I., CONCISE ENCYCLOPEDIA: BIOCHEMISTRY AND MOLECULAR BIOLOGY (de Gruyter, 3d ed. 1997) [hereinafter "Scott, CONCISE ENCYCLOPEDIA”] and Watson, J.D. et al., RECOMBINANT DNA (2d ed. 1992) [hereinafter "Watson, RECOMBINANT DNA”], each of which is inco ⁇ orated herein by reference in its entirety for all pu ⁇ oses.
• immunogen refers to a substance that is capable of provoking an immune response.
• immunogens include, e.g., antigens, autoantigens that play a role in induction of autoimmune diseases, and tumor-associated antigens expressed on cancer cells.
• an "antigen” is a substance that is capable of eliciting the formation of antibodies in a host or generating a specific population of lymphocytes reactive with that substance.
• Antigens are typically macromolecules (e.g., proteins and polysaccharides) that are foreign to the host.
• the term "immunoassay” includes an assay that uses an antibody or immunogen to bind or specifically bind an antigen. The immunoassay is typically characterized by the use of specific binding properties of a particular antibody to isolate, target, and /or quantify the antigen.
• the term “homology” generally refers to the degree of similarity between two or more structures.
• the term “homologous sequences” refers to regions in macromolecules that have a similar order of monomers.
• the term “homology” refers to the degree of similarity between two or more nucleic acid sequences (e.g., genes) or fragments thereof.
• the degree of similarity between two or more nucleic acid sequences refers to the degree of similarity of the composition, order, or arrangement of two or more nucleotide bases (or other genotypic feature) of the two or more nucleic acid sequences.
• homologous nucleic acids generally refers to nucleic acids comprising nucleotide sequences having a degree of similarity in nucleotide base composition, arrangement, or order.
• the two or more nucleic acids may be of the same or different species or group.
• percent homology when used in relation to nucleic acid sequences, refers generally to a percent degree of similarity between the nucleotide sequences of two or more nucleic acids.
• homoology refers to the degree of similarity between two or more polypeptide (or protein) sequences (e.g., genes) or fragments thereof.
• the degree of similarity between two or more polypeptide (or protein) sequences refers to the degree of similarity of the composition, order, or arrangement of two or more amino acid of the two or more polypeptides (or proteins).
• the two or more polypeptides (or proteins) may be of the same or different species or group.
• the term "percent homology” when used in relation to polypeptide (or protein) sequences refers generally to a percent degree of similarity between the amino acid sequences of two or more polypeptide (or protein) sequences.
• homologous polypeptides or “homologous proteins” generally refers to polypeptides or proteins, respectively, that have amino acid sequences and functions that are similar.
• Such homologous polypeptides or proteins may be related by having amino acid sequences and functions that are similar, but are derived or evolved from different or the same species using the techniques described herein.
• subject as used herein includes, but is not limited to, an organism; a mammal, including, e.g., a human, non-human primate (e.g., monkey), mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; a non-mammal, including, e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish; and a non-mammalian invertebrate.
• pharmaceutical composition means a composition suitable for pharmaceutical use in a subject, including an animal or human.
• a pharmaceutical composition generally comprises an effective amount of an active agent and a pharmaceutically acceptable carrier.
• the term "effective amount" means a dosage or amount sufficient to produce a desired result.
• the desired result may comprise an objective or subjective improvement in the recipient of the dosage or amount.
• a prophylactic treatment is a treatment administered to a subject who does not display signs or symptoms of a disease, pathology, or medical disorder, or displays only early signs or symptoms of a disease, pathology, or disorder, such that treatment is administered for the pu ⁇ ose of diminishing, preventing, or decreasing the risk of developing the disease, pathology, or medical disorder.
• a prophylactic treatment functions as a preventative treatment against a disease or disorder.
• a “prophylactic activity” is an activity of an agent, such as a nucleic acid, vector, gene, polypeptide, protein, substance, composition thereof that, when administered to a subject who does not display signs or symptoms of pathology, disease or disorder, or who displays only early signs or symptoms of pathology, disease, or disorder, diminishes, prevents, or decreases the risk of the subject developing a pathology, disease, or disorder.
• a “prophylactically useful” agent or compound refers to an agent or compound that is useful in diminishing, preventing, treating, or decreasing development of pathology, disease or disorder.
• a “therapeutic treatment” is a treatment administered to a subject who displays symptoms or signs of pathology, disease, or disorder, in which treatment is administered to the subject for the pu ⁇ ose of diminishing or eliminating those signs or symptoms of pathology, disease, or disorder.
• a “therapeutic activity” is an activity of an agent, such as a nucleic acid, vector, gene, polypeptide, protein, substance, or composition thereof, that eliminates or diminishes signs or symptoms of pathology, disease or disorder, diminishes when administered to a subject suffering from such signs or symptoms.
• a “therapeutically useful” agent or compound indicates that an agent or compound is useful in diminishing, treating, or eliminating such signs or symptoms of a pathology, disease or disorder.
• Gene broadly refers to any segment of DNA associated with a biological function. Genes include coding sequences and/or regulatory sequences required for their expression. Genes also include non-expressed DNA nucleic acid segments that, e.g., form recognition sequences for other proteins.
• oligonucleotide synthesis and purification steps are performed according to specifications.
• the techniques and procedures are generally performed according to conventional methods in the art and various general references which are provided throughout this document. The procedures therein are believed to be well known to those of ordinary skill in the art and are provided for the convenience of the reader.
• the invention provides isolated or recombinant interferon-alpha homologue polypeptides, and isolated or recombinant polynucleotides encoding the polypeptides.
• polynucleotide sequences which encode novel interferon-alpha homologue polypeptides are collectively referred to herein as "interferon-alpha homologues," "interferon homologue nucleic acids,” “IFN- alpha homologues,” “IFN homologues,” “IFN nucleic acids,” “interferon homologues,” “interferon nucleic acids, “recombinant interferon-alpha,” “recombinant interferon-alpha nucleic acids,” “nucleic acids of the invention,” “polynucleotides of the invention,” or “nucleotides of the invention.”
• Polynucleotide, nucleotide are
• nucleic acid is used interchangeable with the term “nucleotide.”
• Polynucleotides encoding the polypeptides of the invention were discovered in libraries of shuffled interferon-alpha related sequences. The library members were screened for antiproliferative activity against human tumor cell lines and, in some cases, assayed for antiviral activity against virus-infected human cells. A subset of the sequences provided herein were discovered in shuffled libraries screened for antiviral activity against virus-infected mouse cells. Coding sequences for interferon homologues were identified as described in the examples. Briefly, libraries of shuffled mature interferon-alpha coding sequences were introduced into E. coli.
• Colonies were screened in a high-throughput antiproliferative activity assay against a human Daudi tumor cell line as described in Example 1, and colonies expressing active polypeptides were selected, re-screened, and expression levels determined. DNA from selected colonies was isolated and re-shuffled to create secondary libraries. The secondary libraries were introduced into E. coli and screened for antiproliferative activity in the human Daudi cell line-based cell proliferation assay. DNA from colonies selected from the primary and secondary library screens were transduced into Chinese hamster ovary (CHO) cells, and stable cell lines were generated.
• CHO Chinese hamster ovary
• CHO- expressed proteins were purified, quantitated, and assayed for antiproliferative activity using the human Daudi cell line, and optionally, for antiviral activity using encephalomyocarditis virus (EMCV)-infected human WISH cells, as described in Example 1.
• EMCV encephalomyocarditis virus
• Exemplary shuffled nucleic acids which encode interferon-alpha homologue polypeptides having antiproliferative activity in the human Daudi cell line-based assay are identified herein as SEQ ID NO: 1 to SEQ ID NO:35, which encode mature interferon- alpha homologue polypeptides identified herein as SEQ ID NO:36 to SEQ ID NO:70, respectively.
• shuffled mature interferon-alpha coding sequences were also screened in a high-throughput antiviral activity screen against EMCV-infected mouse cells.
• Exemplary shuffled nucleic acids which encode polypeptides having antiviral activity in the murine cell /EMCV-based assay are identified herein as SEQ ID NO:72 to SEQ ED NO:78, which encode mature interferon homologue polypeptides identified herein as SEQ ID NO:79 to SEQ ID NO:85.
• the invention provides an isolated or recombinant nucleic acid that comprises a polynucleotide sequence selected from the group of: (a) SEQ ID NO:l to SEQ ID NO:35, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO:36 to SEQ ID NO:71, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which hybridizes under at least stringent or at least highly stringent hybridization conditions (or ultra-high stringent or ultra-ultra- high stringent hybridization conditions) over substantially the entire length of polynucleotide sequence (a) or (b), or with a 50, 120, 130, 140, 145, 150, 155, 160, or 165 nucleotide base subsequence or fragment of a polynucleotide sequence of (a) or (b); and (d) a polynucle
• the invention provides an isolated or recombinant nucleic acid that comprises a polynucleotide sequence selected from the group of: (a) SEQ ID NO:72 to SEQ ED NO:78, or a complementary polynucleotide sequence thereof; (b) a polynucleotide sequence encoding a polypeptide selected from SEQ ID NO:79 to SEQ ID NO:85, or a complementary polynucleotide sequence thereof; (c) a polynucleotide sequence which hybridizes under at least stringent or at least highly stringent hybridization conditions (or ultra-high stringent or ultra-ultra- high stringent hybridization conditions) over substantially the entire length of polynucleotide sequence (a) or (b), or with a 50, 120, 130, 140, 145, 150, 155, 160, or 165 nucleotide base subsequence or fragment of a polynucleotide sequence of (a) or (b); and (d) a
• the invention also includes an isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises the amino acid sequence: CDLPQTHSLG-X ⁇ -X 12 -RA-Xi 5 -Xi 6 -LL-Xi 9 -QM- X 22 -R-X 24 -S-X 26 -FSCLKDR-X 34 -DFG-X 38 -P-X 4 o-EEFD-X 45 -X 46 -X 47 -FQ-X 5 o-X 51 -QAI- X 55 -X 56 -X 57 -HE-X 60 -X 61 -QQTFN-X 67 -FSTK-X 72 -SS-X 75 -X 76 -W-X 78 -X 79 -X 80 -LL-X 83 -K- X 85 -X 86 -T-X 88 -L-X 9
• polypeptides having an antiproliferative activity in the human Daudi cell line-based assay e.g., at least about 8.3xl0 6 units/mg
• an antiviral activities in a human WISH cell/EMCV-based assay at least about 2.1xl0 7 units/mg
• the polynucleotides of the invention are useful in for a variety of applications, including, but not limited to, as therapeutic and prophylactic agents in methods of in vivo and ex vivo treatment of a variety of diseases, disorders, and conditions in a variety of subjects; for use in in vitro methods, such as diagnostic methods, to detect, diagnose, and treat a variety of diseases, disorders, and conditions in a variety of subjects; for use in, e.g., gene therapy; as therapeutics and prophylactics, e.g., for use in methods of therapeutic and prophylactic treatment of a disease, disorder or condition; as immunogens; for use in diagnostic and screening assays; and as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of IFN-alpha coding nucleic acids).
• Polynucleotides and oligonucleotides of the invention can be prepared by standard solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 20, 30, 40, 50, 60, 70, 80, 90, and/or 100 nucleotide bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase mediated recombination methods) to form essentially any desired continuous sequence.
• nucleotide fragments of greater than 100 nucleotide bases are individually synthesized, then joined (e.g., by enzymatic or chemical ligation methods, or polymerase mediated recombination methods) to form essentially any desired continuous sequence
• the polynucleotides and oligonucleotides of the invention, including fragments thereof (and those as described herein) can be prepared by chemical synthesis using, e.g., the classical phosphoramidite method described by Beaucage et al.
• oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, li gated and cloned in appropriate vectors.
• nucleic acid can be custom ordered from any of a variety of commercial sources, such as The Midland Certified Reagent Company (mcrc@oligos.com), The Great American Gene Company (http://www.genco.com), ExpressGen Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda, CA) and many others.
• peptides and antibodies can be custom ordered from any of a variety of sources, such as PeptidoGenic (pkim@ccnet.com), HTI Bio-products, inc. (http://www.htibio.com), BMA Biomedicals Ltd. (U.K.), Bio. Synthesis, Inc., and many others.
• Certain polynucleotides of the invention may also obtained by screening cDNA libraries (e.g., libraries generated by recombining homologous nucleic acids as in typical diversity generation methods, such as, e.g., shuffling methods) using oligonucleotide probes which can hybridize to or PCR-amplify polynucleotides which encode the interferon homologue polypeptides and fragments of those polypeptides.
• cDNA libraries e.g., libraries generated by recombining homologous nucleic acids as in typical diversity generation methods, such as, e.g., shuffling methods
• oligonucleotide probes which can hybridize to or PCR-amplify polynucleotides which encode the interferon homologue polypeptides and fragments of those polypeptides.
• the polynucleotides of the invention include sequences which encode novel mature interferon-alpha homologues and sequences complementary to the coding sequences, and novel fragments of such coding sequences and complements thereof.
• the polynucleotides can be in the form of RNA or in the form of DNA, and include mRNA, cRNA, synthetic RNA and DNA, and cDNA.
• the polynucleotides can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding (anti-sense, complementary) strand.
• the polynucleotides optionally include the coding sequence of an interferon-alpha homologue (i) in isolation, (ii) in combination with additional coding sequence, so as to encode, e.g., a fusion protein, a pre-protein, a prepro-protein, or the like, (iii) in combination with non- coding sequences, such as introns, control elements such as a promoter, a terminator element, or 5' and/or 3' untranslated regions effective for expression of the coding sequence in a suitable host, and/or (iv) in a vector or host environment in which the interferon-alpha homologue coding sequence is a heterologous nucleic acid sequence or gene. Sequences can also be found in combination with typical compositional formulations of nucleic acids, including in the presence of carriers, buffers, adjuvants, excipients and the like.
• DNA or RNA encoding the respective interferon-alpha homologue polypeptide includes any oligodeoxynucleotide or oligodeoxyribonucleotide sequence which, upon expression in an appropriate host cell, results in production of an interferon-alpha homologue polypeptide of the invention.
• the DNA or RNA can be produced in an appropriate host cell, or in a cell-free (in vitro) system, or can be produced synthetically (e.g., by an amplification technique such as PCR) or chemically.
• the polynucleotides of the invention have a variety of uses in, for example: recombinant production (i.e., expression) of the interferon-alpha homologue polypeptides of the invention; as therapeutics and prophylactics, e.g., for use in methods of therapeutic and prophylactic treatment of a disease, disorder or condition; for use in, gene therapy methods and related applications;; as immunogens; for use in diagnostic and screening assays; as diagnostic probes for the presence of complementary or partially complementary nucleic acids (including for detection of natural EFN- alpha coding nucleic acids); as substrates for further reactions, e.g., shuffling reactions or mutation reactions to produce new and/or improved IFN-alpha homologues, and the like.
• interferon-alpha homologue polypeptides which encode novel and/or mature interferon-alpha homologues, fragments of interferon-alpha proteins, related fusion proteins, or functional equivalents thereof, are collectively referred to herein as "interferon-alpha homologue polypeptides," "interferon-alpha homologue proteins,” or “interferon-alpha homologues,” "interferon homologues,” “IFN-alpha homologues,” “EFN homologues”, “IFN polypeptides,” “IFN proteins” "polypeptides of the invention,” or “proteins of the invention.” Polypeptide or amino acid fragments of each of the preceding terms are also intended to be included and encompassed in the polypeptides or proteins of the invention.
• Such polynucleotide sequences of the invention are used in recombinant DNA (or RNA) molecules that direct the expression of the interferon-alpha homologue polypeptides in appropriate host cells. Due to the inherent degeneracy of the genetic code, other nucleic acid sequences which encode substantially the same or a functionally equivalent amino acid sequence are also used to clone and express the interferon homologues.
• a coding sequence including, e.g., a nucleotide sequence encoding an interferon- alpha homologue of the invention or a fragment thereof
• the genetic code is redundant with 64 possible codons, but most organisms preferentially use a subset of these codons.
• the codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons (see, e.g., Zhang S.P. et al. (1991) Gene 105:61-72). Codons can be substituted to reflect the preferred codon usage of the host, a process called "codon optimization" or "controlling for species codon bias.”
• Optimized coding sequence containing codons prefened by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
• Translation stop codons can also be modified to reflect host preference. For example, preferred stop codons for 5. cerevisiae and mammals are UAA and UGA, respectively. The preferred stop codon for monocotyledonous plants is UGA, whereas insects and E.
• polynucleotide sequences of the present invention can be engineered in order to alter an interferon homologue coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.
• alterations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to introduce splice sites, etc.
• the present invention also includes recombinant constructs comprising one or more of the nucleic acid sequences as broadly described herein (e.g., those encoding an interferon-alpha homologue of the invention or a fragment thereof).
• the constructs comprise a vector, such as, a plasmid, a cosmid, a phage, a virus (including a retrovirus), a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and the like, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation.
• the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
• regulatory sequences including, for example, a promoter, operably linked to the sequence.
• suitable vectors and promoters are known to those of skill in the art, and are commercially available.
• General texts which describe molecular biological techniques useful herein, including the use of vectors, promoters and many other relevant topics, include Juo, P-S., CONCISE DICTIONARY OF BIOMEDICAL AND MOLECULAR BIOLOGY (CRC Press 1996); Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed.
• RNA polymerase mediated techniques e.g., NASBA
• PCR polymerase chain reaction
• LCR ligase chain reaction
• NASBA RNA polymerase mediated techniques
• PCR generally refers to a procedure wherein minute amounts of a specific piece of nucleic acid, RNA, and/or DNA, are amplified by methods well known in the art (see, e.g., U.S. Pat. No. 4,683,195 and other references above). Generally, sequence information from the ends of the region of interest or beyond is used, for design of oligonucleotide primers. Such primers will be identical or similar in sequence to the opposite strands of the template to be amplified. The 5' terminal nucleotides of the opposite strands may coincide with the ends of the amplified material.
• PCR may be used to amplify specific RNA or specific DNA sequences, recombinant DNA or RNA sequences, DNA and RNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc.
• PCR is one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample comprising the use of a another (e.g., known) nucleic acid as a primer, improved methods of cloning in vitro amplified nucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.
• RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase. See Ausubel, Sambrook and Berger, all supra.
• the present invention also relates to host cells which are transduced with vectors of the invention, and the production of polypeptides of the invention (including fragments thereof) by recombinant techniques.
• Host cells are genetically engineered (i.e., transduced, transformed or transfected) with the vectors of this invention, which may be, for example, a cloning vector or an expression vector.
• the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
• the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the interferon homologue gene.
• the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art and in the references cited herein, including, e.g., Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3d ed., Wiley-Liss, New York and the references cited therein.
• interferon homologue polypeptides and proteins of the invention can also be produced in non-animal cells such as plants, yeast, fungi, bacteria and the like.
• non-animal cells such as plants, yeast, fungi, bacteria and the like.
• details regarding cell culture can be found in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods, Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL.
• the polynucleotides of the present invention may be included in any one of a variety of expression vectors for expressing a polypeptide.
• Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies, adenovirus, adeno-associated virus, retroviruses and many others.
• the nucleic acid sequence in the expression vector is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis.
• promoters include: LTR or SV40 promoter, E. coli lac or t ⁇ promoter, phage lambda P promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
• the expression vector also contains a ribosome binding site for translation initiation, and a transcription terminator.
• the vector optionally includes appropriate sequences for amplifying expression.
• the expression vectors optionally comprise one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
• selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
• the vector containing the appropriate DNA sequence as described herein, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
• appropriate expression hosts include: bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium; fungal cells, such as Saccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insect cells such as Drosophila and Spodoptera frugiperda; mammalian cells such as CHO, COS, BHK, HEK 293 or Bowes melanoma; plant cells, etc.
• interferon homologues not all cells or cell lines need to be capable of producing fully functional interferon homologues; for example, antigenic fragments of an interferon homologue may be produced in a bacterial or other expression system.
• the invention is not limited by the host cells employed.
• a number of expression vectors may be selected depending upon the use intended for the interferon homologue. For example, when large quantities of interferon homologue or fragments thereof are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable.
• Such vectors include, but are not limited to, multifunctional E.
• coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the interferon homologue coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster (1989) J. Biol. Chem. 264:5503-5509); pET vectors (Novagen, Madison WI); and the like.
• BLUESCRIPT Stratagene
• pIN vectors Van Heeke & Schuster (1989) J. Biol. Chem. 264:5503-5509
• pET vectors Novagen, Madison WI
• yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used for production of the interferon homologue proteins of the invention.
• constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH
• PGH interferon homologue proteins of the invention.
• a number expression systems such as viral-based systems, may be utilized.
• a coding sequence is optionally ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence.
• transcription enhancers such as the rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
• RSV rous sarcoma virus
• Specific initiation signals can aid in efficient translation of an interferon homologue coding sequence. These signals can include, e.g., the ATG initiation codon and adjacent sequences. In cases where interferon homologue coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence (e.g., a mature protein coding sequence), or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the co ⁇ ect reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic.
• Polynucleotides of the invention can also be fused, for example, in-frame to nucleic acid encoding a secretion/localization sequence, to target polypeptide expression to a desired cellular compartment, membrane, or organelle, or to direct polypeptide secretion to the periplasmic space or into the cell culture media.
• sequences are known to those of skill, and include secretion leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, ER retention signals, mitochondrial transit sequences, chloroplast transit sequences), membrane localization/anchor sequences (e.g., stop transfer sequences, GPI anchor sequences), and the like.
• Polypeptides expressed by such polynucleotides of the invention may include the amino acid sequence corresponding to the secretion and/or localization sequence(s).
• the present invention relates to host cells containing the above-described constructs.
• the host cell can be a eukaryotic cell, such as a mammalian cell, a yeast cell, or a plant cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
• Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, or other common techniques (Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in
• the cell may include a nucleic acid of the invention, said nucleic acid encoding a polypeptide, wherein said cells expresses a polypeptide (e.g., an interferon-alpha homologue polypeptide having an antiviral or anti-proliferative activity as measured by the assays described herein).
• the invention also includes a vector comprising any nucleic acid of the invention described herein and includes a cell transduced by such a vector.
• Cells and transgenic animals which include any polypeptide or nucleic acid above or throughout this specification, e.g., produced by transduction of a vector of the invention, are an additional feature of the invention.
• a host cell strain is optionally chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
• modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
• Post-translational processing which cleaves a "pre” or a "prepro” form of the protein may also be important for correct insertion, folding and/or function.
• Different host cells such as CHO, HeLa, BHK, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
• stable expression For long-term, high-yield production of recombinant proteins, stable expression can be used.
• cell lines which stably express a polypeptide of the invention are transduced using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
• the pu ⁇ ose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
• resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
• Host cells transformed with a nucleotide sequence encoding a polypeptide of the invention are optionally cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
• the protein or fragment thereof produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly, depending on the sequence and/or the vector used.
• expression vectors containing polynucleotides encoding mature interferon homologues of the invention can be designed with signal sequences which direct secretion of the mature polypeptides through a prokaryotic or eukaryotic cell membrane.
• the polynucleotides of the present invention may also comprise a coding sequence fused in-frame to a marker sequence which, e.g., facilitates purification of the encoded polypeptide of the invention.
• purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, a sequence which binds glutathione (e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitope derived from the influenza hemagglutinin protein; Wilson, I. et al.
• One expression vector contemplated for use in the compositions and methods described herein provides for expression of a fusion protein comprising a polypeptide of the invention fused to a polyhistidine region separated by an enterokinase cleavage site. The histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath et al.
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
• GST glutathione S-transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion to ligand-agarose beads (e.g., glutathione-agarose in the case of GST- fusions) followed by elution in the presence of free ligand.
• the selected promoter is induced by appropriate means
• Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art.
• Polypeptides of the invention can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using any of the tagging systems noted herein), hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as desired, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification steps.
• HPLC high performance liquid chromatography
• Modified Amino Acids Polypeptides of the invention may contain one or more modified amino acids. The presence of modified amino acids may be advantageous in, for example, (a) increasing polypeptide serum half-life, (b) reducing polypeptide antigenicity, (c) increasing polypeptide storage stability.
• Amino acid(s) are modified, for example, co- translationally or post-translationally during recombinant production (e.g., N-linked glycosylation at N-X-S/T motifs during expression in mammalian cells) or modified by synthetic means.
• a modified amino acid include a glycosylated amino acid, a sulfated amino acid, a prenylated (e.g., farnesylated, geranylgeranylated) amino acid, an acetylated amino acid, an acylated amino acid, a PEG-ylated amino acid, a biotinylated amino acid, a carboxylated amino acid, a phosphorylated amino acid, and the like.
• References adequate to guide one of skill in the modification of amino acids are replete throughout the literature. Example protocols are found in Walker (1998) Protein Protocols on CD-ROM Human Press, Towata, NJ.
• polynucleotides and polypeptides of the invention have a variety of uses, including, but not limited to, for example: in recombinant production (i.e., expression) of the recombinant interferon-alpha homologues of the invention; as therapeutic and prophylactic agents in methods of in vivo and ex vivo treatment of a variety of diseases, disorders, and conditions in a variety of subjects; for use in in vitro methods, such as diagnostic and screening methods, to detect, diagnose, and treat a variety of diseases, disorders, and conditions (e.g., cancers, viral -based disorders, angiogenic- based disorders) in a variety of subjects (e.g., mammals); as immunogens; in gene therapy methods and DNA- or RNA-based delivery methods to deliver or administer in vivo, ex vivo, or in vitro biologically active polypeptides of the invention to a tissue, population or cells, organ, graft, bodily system of a subject (e.g., organ system, lymph
• Polynucleotides which encode an interferon homologue of the invention, or complements of the polynucleotides, are optionally administered to a cell to accomplish a therapeutically or prophylactically useful process or to express a therapeutically useful product in vivo, ex vivo, or in vitro.
• These applications, including in vivo or ex vivo applications, including, e.g., gene therapy, include a multitude of techniques by which gene expression may be altered in cells. Such methods include, for instance, the introduction of genes for expression of, e.g., therapeutically or prophylactically useful polypeptides, such as the interferon homologues of the present invention.
• retrovirus comprising the polynucleotides and/or polypeptides of the invention.
• retrovirus further comprises additional exogenous, e.g., therapeutic or prophylactic gene construct, sequences.
• the invention provides gene therapy methods of prophylactically or therapeutically treating a disease, disorder or condition in a subject in need of such treatment by administering in vivo, ex vivo, or in vitro one or more nucleic acids of the invention described herein to one or more cells of a subject, including an organism or mammal, including, e.g., a human, primate, mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, sheep; or a non-mammalian vertebrate such as a bird (e.g., a chicken or duck) or a fish, or invertebrate, as described in more detail below.
• a bird e.g., a chicken or duck
• fish or invertebrate
• the invention provides methods of prophylactically or therapeutically treating a disease, disorder or condition in a subject in need of such treatment by administering in vivo, ex vivo, or in vitro one or more polypeptides of the invention described herein to one or more cells of a subject (including those defined herein), as described in more detail below.
• Polynucleotides encoding interferon homologue polypeptides of the invention are particularly useful for in vivo or ex vivo therapeutic or prophylactic applications, using techniques well known to those skilled in the art.
• cultured cells are engineered ex vivo with a polynucleotide (DNA or RNA), with the engineered cells then being returned to the patient.
• Cells may also be engineered in vivo or ex vivo for expression of a polypeptide in vivo or ex vivo, respectively.
• a number of viral vectors suitable for organismal in vivo or ex vivo transduction and expression are known. Such vectors include retroviral vectors (see Miller(1992) Curr. Top. Microbiol. Immunol.
• Gene therapy provides methods for combating chronic infectious diseases (e.g., HIV infection, viral hepatitis, He ⁇ es Simplex Virus (HSV), hepatitis B (HepB), dengue virus, etc.), as well as non-infectious diseases including cancer and allergic diseases and some forms of congenital defects such as enzyme deficiencies.
• chronic infectious diseases e.g., HIV infection, viral hepatitis, He ⁇ es Simplex Virus (HSV), hepatitis B (HepB), dengue virus, etc.
• HSV He ⁇ es Simplex Virus
• HepB hepatitis B
• dengue virus etc.
• nucleic acids include liposome based gene delivery (Debs and Zhu (1993) WO 93/24640 and U.S. Pat. No. 5,641,662; Mannino and Gould-Fogerite (1988) BioTechniques 6(7):682-691; Rose, U.S. Pat No.
• adenoviral vector mediated gene delivery e.g., to treat cancer (.see, e.g., Chen et al. (1994) Proc. Nat'lAcad. Sci. C/SA . 91:3054-3057; Tong et al. (1996) Gynecol. Oncol. 61:175-179; Clayman et al. (1995) Cancer Res. 5:1-6; O'Malley et al. (1995) Cancer Res. 55: 1080-1085; Hwang et al. (1995) Am. J. Respir. Cell Mol. Biol. 13:7-16; Haddada et al. (1995) Curr. Top. Microbiol. Immunol.
• nucleic acids of the invention are also useful for sense and anti-sense suppression of expression, e.g., to down-regulate expression of a nucleic acid of the invention, once expression of the nucleic acid is no-longer desired in the cell.
• nucleic acids of the invention, or subsequences or anti-sense sequences thereof can also be used to block expression of naturally occurring homologous nucleic acids.
• sense and anti-sense technologies are known in the art, e.g., as set forth in Lichtenstein and Nellen (1997) Antisense Technology: A Practical Approach IRL Press at
• polynucleotides and polypeptides of the invention may be employed for therapeutic and prophylactic uses in combination with a suitable pharmaceutical carrier.
• suitable pharmaceutical carrier comprise a therapeutically or prophylactically effective amount of the polynucleotide or polypeptide of the invention, and a pharmaceutically acceptable carrier or excipient.
• a pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, buffers and excipients.
• Such a carrier or excipient includes, but is not limited to, saline, buffered saline (e.g., phosphate- buffered saline solution), dextrose, water, glycerol, ethanol, emulsions (such as an oil/water or water/oil emulsion), various types of wetting agents and/or adjuvants, and combinations thereof.
• saline e.g., phosphate- buffered saline solution
• dextrose e.g., phosphate- buffered saline solution
• water glycerol
• ethanol emulsions
• wetting agents and/or adjuvants emulsions
• Suitable pharmaceutical carriers and agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Publishing Co., Easton, 19 th ed. 1995).
• the formulation should suit the mode of administration of the active agent (e.g., nucleo
• polynucleotides also referred to herein as oligonucleotides, typically having at least 12 bases, preferably at least 15, more preferably at least 20, 30, or 50 bases, which hybridize under at least highly stringent (or ultra-high stringent or ultra-ultra- high stringent conditions) conditions to an interferon homologue polynucleotide sequence described above.
• the polynucleotides may be used as probes, primers, sense and antisense agents, and the like, according to methods as noted supra.
• nucleic acids sequences encoding interferon homologue polypeptides of the invention may be produced, some which may bear minimal sequence homology to the nucleic acid sequences explicitly disclosed herein.
• AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acid arginine.
• the codon can be altered to any of the conesponding codons described above without altering the encoded polypeptide. It is understood that U in an RNA sequence corresponds to T in a DNA sequence.
• a silent variation of this sequence includes TGC GAC TTA CCA CAA, both sequences which encode the amino acid sequence CDLPQ, corresponding to amino acids 1-5 of SEQ ID NO:36.
• Such "silent variations" are one species of “conservatively modified variations,” discussed below.
• each codon in a nucleic acid can be modified by standard techniques to encode a functionally identical polypeptide. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in any described sequence.
• the invention provides each and every possible variation of nucleic acid sequence encoding a polypeptide of the invention that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code (e.g., as set forth in Table 1) as applied to the nucleic acid sequence encoding an interferon homologue polypeptide of the invention. All such variations of every nucleic acid herein are specifically provided and described by consideration of the sequence in combination with the genetic code.
• Constantly modified variations or, simply, “conservative variations” of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
• substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 3%, 2% or 1%) in an encoded sequence are "conservatively modified variations” where the alterations result in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid.
• “conservatively substituted variations” or “conservative substitutions” of a listed polypeptide sequence of the present invention include substitutions of a small percentage, typically less than 5%, more typically less than 4%, 3%, 2% or 1%, of the amino acids of the polypeptide sequence, with a conservatively selected amino acid of the same conservative substitution group.
• a conservatively substituted variation of the polypeptide identified herein as SEQ ID NO:36 will contain "conservative substitutions", according to the six groups defined above, in up to about 8 or 9 residues (i.e., about 5% of the amino acids) in the 166-amino acid polypeptide.
• WEVVR AEIMR SFSFS TNLQK RLRRKE include: WEVVR SEIMR SFS YS TNLQR RLRRKD and
• nucleic acid molecule which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional sequence, is a conservative variation of the basic nucleic acid.
• nucleic acid constructs which are disclosed yield a functionally identical construct.
• silent substitutions i.e., substitutions in a nucleic acid sequence which do not result in an alteration in an encoded polypeptide
• conserve amino acid substitutions in one or a few amino acids in an amino acid sequence are substituted with different amino acids with highly similar properties, are also readily identified as being highly similar to a disclosed construct.
• conservative variations of each disclosed sequence are a feature of the present invention.
• nucleic acids hybridize due to a variety of well characterized physico-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory
• highly stringent hybridization and wash conditions are selected to be about 5° C or less lower lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH (as noted below, highly stringent conditions can also be referred to in comparative terms).
• T m is the temperature (under defined ionic strength and pH) at which 50% of the test sequence hybridizes to a perfectly matched probe.
• Very stringent conditions are selected to be equal to the T m for a particular probe.
• the T m is the temperature of the nucleic acid duplexes indicates the temperature at which the duplex is 50% denatured under the given conditions and its represents a direct measure of the stability of the nucleic acid hybrid.
• the T m corresponds to the temperature corresponding to the midpoint in transition from helix to random coil; it depends on length, nucleotide composition, and ionic strength for long stretches of nucleotides.
• unhybridized nucleic acid material can be removed by a series of washes, the stringency of which can be adjusted depending upon the desired results. Low stringency washing conditions (e.g., using higher salt and lower temperature) increase sensitivity, but can product nonspecific hybridization signals and high background signals.
• T m (°C) 81.5°C + 16.6 (log, 0 M) + 0.41 (%G + C) - 0.72 (%f) - 500/n, where M is the molarity of the monovalent cations (usually Na+), (%G + C) is the percentage of guanosine (G) and cystosine (C ) nucleotides, (%f) is the percentage of formalize and n is the number of nucleotide bases (i.e. , length) of the hybrid. See Rapley and Walker, supra.
• the T m of an RNA-DNA duplex can be estimated as follows:
• T m (°C) 79.8°C + 18.5 (log ⁇ 0 M) + 0.58 (%G + C) - 11.8(%G + C) 2 - 0.56 (%f) - 820/n, where M is the molarity of the monovalent cations (usually Na+), (%G + C)is the percentage of guanosine (G ) and cystosine (C ) nucleotides, (%f) is the percentage of formamide and n is the number of nucleotide bases (i.e., length) of the hybrid. Id.
• Equations 1 and 2 are typically accurate only for hybrid duplexes longer than about 100-200 nucleotides. Id.
• the Tm of nucleic acid sequences shorter than 50 nucleotides can be calculated as follows:
• T ra (°C) 4(G + C) + 2(A + T), where A (adenine), C, T (thymine), and G are the numbers of the conesponding nucleotides.
• An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42°C, with the hybridization being carried out overnight.
• An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see Sambrook, supra for a description of SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove background probe signal.
• An example low stringency wash is 2x SSC at 40°C for 15 minutes.
• a signal to noise ratio of 2.5x-5x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
• Detection of at least stringent hybridization between two sequences in the context of the present invention indicates relatively strong structural similarity or homology to, e.g., the nucleic acids of the present invention provided in the sequence listings herein.
• highly stringent conditions are selected to be about 5° C or less lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
• Target sequences that are closely related or identical to the nucleotide sequence of interest e.g., "probe”
• T m thermal melting point
• Lower stringency conditions are appropriate for sequences that are less complementary. See, e.g., Rapley and Walker, supra.
• Comparative hybridization can be used to identify nucleic acids of the invention, and this comparative hybridization method is a preferred method of distinguishing nucleic acids of the invention.
• Detection of highly stringent hybridization between two nucleotide sequences in the context of the present invention indicates relatively strong structural similarity/homology to, e.g., the nucleic acids provided in the sequence listing herein.
• Highly stringent hybridization between two nucleotide sequences demonstrates a degree of similarity or homology of structure, nucleotide base composition, arrangement or order that is greater than that detected by stringent hybridization conditions.
• detection of highly stringent hybridization in the context of the present invention indicates strong structural similarity or structural homology (e.g., nucleotide structure, base composition, a ⁇ angement or order) to, e.g., the nucleic acids provided in the sequence listings herein.
• structural similarity or structural homology e.g., nucleotide structure, base composition, a ⁇ angement or order
• one measure of stringent hybridization is the ability to hybridize to one of the listed nucleic acids (e.g., nucleic acid sequences SEQ ID NO:l to SEQ ID NO:35, and SEQ ID NO:72 to SEQ ED NO:78, and complementary polynucleotide sequences thereof) under highly stringent conditions (or very stringent conditions, or ultra- high stringency hybridization conditions, or ultra-ultra high stringency hybridization conditions).
• Stringent hybridization including, e.g., highly stringent, ultra-high stringency, or ultra-ultra high stringency hybridization conditions
• wash conditions can easily be determined empirically for any test nucleic acid.
• the hybridization and wash conditions are gradually increased (e.g., by increasing temperature, decreasing salt concentration, increasing detergent concentration and/or increasing the concentration of organic solvents, such as formalin, in the hybridization or wash), until a selected set of criteria are met.
• the hybridization and wash conditions are gradually increased until a probe comprising one or more nucleic acid sequences selected from SEQ ID NO: 1 to SEQ ED NO:35, SEQ ED
• nucleic acid comprising one or more nucleic acid sequences selected from SEQ ID NO:l to SEQ ID NO:35, SEQ ID NO:72 to SEQ ID NO:78, and complementary polynucleotide sequences thereof, with a signal to noise ratio that is at least 2.5x, and optionally 5x or more as high as that observed for hybridization of the probe to an unmatched target.
• the unmatched target is a nucleic acid corresponding to a known alpha interferon, e.g., an alpha interferon nucleic acid that is present in a public database such as GenBankTM at the time of filing of the subject application.
• a known alpha interferon e.g., an alpha interferon nucleic acid that is present in a public database such as GenBankTM at the time of filing of the subject application.
• Examples of such unmatched target nucleic acids include, e.g., those with the following GenBank accession numbers: J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), R0067 (Gx-1), 101614, 101787, 107821, M12350 (alpha-F), M38289, V00549 (alpha-2a), and 108313 (alpha-Conl).
• a test nucleic acid is said to specifically hybridize to a probe nucleic acid when it hybridizes at least Vi as well to the probe as to the perfectly matched complementary target, i.e., with a signal to noise ratio at least Vi as high as hybridization of the probe to the target under conditions in which the perfectly matched probe binds to the perfectly matched complementary target with a signal to noise ratio that is at least about 2.5x-10x, typically 5x-10x as high as that observed for hybridization to any of the unmatched target nucleic acids represented by GenBank accession numbers J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02
• Ultra high-stringency hybridization and wash conditions are those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least lOx as high as that observed for hybridization to any of the unmatched target nucleic acids represented by GenBank accession numbers J00210 (alpha-D), J00207
• even higher levels of stringency can be determined by gradually increasing the hybridization and/or wash conditions of the relevant hybridization assay. For example, those in which the stringency of hybridization and wash conditions are increased until the signal to noise ratio for binding of the probe to the perfectly matched complementary target nucleic acid is at least lOx, 20X, 50X, 100X, or 500X or more as high as that observed for hybridization to any of the unmatched target nucleic acids represented by GenBank accession numbers J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene),
• Target nucleic acids which hybridize to the nucleic acids represented by SEQ ID ⁇ O:l to SEQ ID NO:35 and SEQ ID NO:72 to SEQ ID NO:78 under high, ultra- high and ultra-ultra high stringency conditions are a feature of the invention.
• nucleic acids include those with one or a few silent or conservative nucleic acid substitutions as compared to a given nucleic acid sequence.
• Nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code, or when antisera or antiserum generated against one or more of SEQ ID NO: 36 to SEQ ID NO: 70 and SEQ ID NO: 79 to SEQ ED NO: 85 which has been subtracted using the polypeptides encoded by known interferon-alpha sequences, including, e.g., the those encoded by the following interferon- alpha nucleic acid sequences in GenBank: Accession numbers J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-IB), X03125 (alpha-8), X0
• TMAC1 hybridization procedure known to those of ordinary skill in the art can be used. See, e.g., Sorg, U. et al. 1 Nucleic Acids Res. (Sept. 11, 1991) 19(17), inco ⁇ orated herein by reference in its entirety for all pu ⁇ oses.
• the invention provides a nucleic acid which comprises a unique subsequence in a nucleic acid selected from SEQ ID NO:l to SEQ ID NO:35 or SEQ ID NO:72 to SEQ ID NO:78.
• the unique subsequence is unique as compared to a nucleic acid corresponding to any known interferon-alpha nucleic acid sequence including, e.g., the known sequences represented by GenBank accession numbers J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (EFN-IB), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), A12109
• Such unique subsequences can be determined by aligning any of SEQ ID NO:l to SEQ ID NO:35 or SEQ ID NO:72 to SEQ ID NO:78 against the complete set of nucleic acids corresponding to GenBank accession numbers of known interferon-alpha nucleic acid sequences, such as, e.g., J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), A12109 (alpha-4B), R0067 (Gx-1), 101614, 101787, 107821, M12350 (
• the invention includes a polypeptide which comprises a unique amino acid subsequence in a polypeptide selected from: SEQ ID NO:36 to SEQ ID NO:70 or SEQ ID NO:79 to SEQ ID NO:85.
• the unique subsequence is unique as compared to an amino acid subsequence of a known interferon-alpha polypeptide including, e.g., an amino acid subsequence of a polypeptide encoded by a known interferon-alpha nucleic acid corresponding to any of GenBank accession numbers: J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), R0067 (Gx-1), 101614, 101787, 107821, M12350 (alpha-F), M38289
• the polypeptide is aligned against the complete set of known interferon-alpha polypeptide sequences, such as those polypeptides encoded by nucleic acids corresponding to GenBank accession numbers J00210 (alpha-D), J00207 (Alpha-A), X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-IB), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), R0067 (Gx-1), 101614, 101787, 107821, M12350 (alpha- F), - M38289N00549 (alpha-2a), and 108313 (alpha-Conl), (referred to as "the control polypeptide
• the present invention provides a target nucleic acid which hybridizes under at least stringent or highly stringent conditions (or conditions of greater stringency) to a unique coding oligonucleotide which encodes a unique subsequence in a polypeptide selected from: SEQ ID NO:36 to SEQ ED NO:70 or SEQ ED NO:79 to SEQ ED NO: 85, wherein the unique subsequence is unique as compared to a an amino acid subsequence of a known interferon-alpha polypeptide sequence shown in GenBank or to a polypeptide conesponding to any of the control polypeptides. Unique sequences are determined as noted above.
• the stringent conditions are selected such that a perfectly complementary oligonucleotide to the coding oligonucleotide hybridizes to the coding oligonucleotide with at least about a 5-10x higher signal to noise ratio than for hybridization of the perfectly complementary oligonucleotide to a control nucleic acid conesponding to any of the control polypeptides.
• Conditions can be selected such that higher ratios of signal to noise are observed in the particular assay which is used, e.g., about 15x, 20x, 30x, 50x or more.
• the target nucleic acid hybridizes to the unique coding oligonucleotide with at least a 2x higher signal to noise ratio as compared to hybridization of the control nucleic acid to the coding oligonucleotide.
• higher signal to noise ratios can be selected, e.g., about 2.5x, about 5x, about lOx, about 20x, about 30x, about 50x or more.
• the particular signal will depend on the label used in the relevant assay, e.g., a fluorescent label, a colorimetric label, a radio active label, or the like.
• the invention provides a polypeptide that comprises unique subsequence in a polypeptide selected from SEQ ID NO:36 to SEQ ID NO:70 and SEQ ID NO:79 to SEQ ID NO:85, wherein the unique subsequence is unique as compared to a polypeptide sequence conesponding to a known interferon-alpha polypeptide, such as, e.g., an interferon-alpha polypeptide sequence present in GenBank.
• the polynucleotides of the invention are useful as substrates for a variety of recombination and recursive recombination (e.g., DNA shuffling) reactions, as well as other diversity generating techniques, including mutagenesis techniques and standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, supra, i.e., to produce additional interferon-alpha homologues with desired properties.
• recombination and recursive recombination e.g., DNA shuffling
• other diversity generating techniques including mutagenesis techniques and standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, supra, i.e., to produce additional interferon-alpha homologues with desired properties.
• recombinant e.g., shuffled, interferon-alpha homologue polypeptides
• recombinant, e.g., shuffled, interferon-alpha homologue polypeptides can be generated and isolated that confer a variety of desirable characteristics, e.g., enhanced antiviral activity, enhanced antiproliferative activity, increased growth inhibitory, cytostatic and/or cytotoxic activities towards particular target cells, reduced immunogenicity, etc.
• a variety of diversity generating protocols including nucleic acid shuffling protocols, are available and fully described in the art.
• the procedures can be used separately, and/or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well variants of encoded proteins. Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified nucleic acids and sets of nucleic acids (including, e.g., nucleic acid libraries) useful, e.g., for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells and/or organisms with new and/or improved characteristics.
• nucleic acids can be selected or screened for nucleic acids that encode proteins with or which confer desirable properties.
• any nucleic acids that are produced can be selected for a desired activity or property, e.g., enhanced antiviral activity, enhanced antiproliferative activity, enhanced anti-angiogenic activity, increased growth inhibitory, cytostatic and/or cytotoxic activities towards particular target cells, reduced immunogenicity, etc.
• Methods for determining nucleic acids having enhanced antiviral, antiproliferative, growth inhibitory, cytostatic, and/or cytotoxic activity or reduced immunogenicity include those described herein. This can include identifying any activity that can be detected, for example, in an automated or automatable format, by any of the assays in the art. A variety of related (or even unrelated) properties can be evaluated, in serial or in parallel, at the discretion of the practitioner.
• nucleic acids can be recombined in vitro by any of a variety of techniques discussed in the references above, including e.g., DNAse digestion of nucleic acids to be recombined followed by ligation and/or PCR reassembly of the nucleic acids.
• nucleic acids can be recursively recombined in vivo or ex vivo, e.g., by allowing recombination to occur between nucleic acids in cells.
• whole genome recombination methods can be used in which whole genomes of cells or other organisms are recombined, optionally including spiking of the genomic recombination mixtures with desired library components (e.g., genes conesponding to the pathways of the present invention).
• oligonucleotides conesponding to targets of interest are synthesized and reassembled in PCR or ligation reactions which include oligonucleotides which conespond to more than one parental nucleic acid, thereby generating new recombined nucleic acids.
• Oligonucleotides can be made by standard nucleotide addition methods, or can be made, e.g., by tri-nucleotide synthetic approaches.
• Fifth, in silico methods of recombination can be effected in which genetic algorithms are used in a computer to recombine sequence strings which conespond to homologous (or even non-homologous) nucleic acids.
• the resulting recombined sequence strings are optionally converted into nucleic acids by synthesis of nucleic acids which conespond to the recombined sequences, e.g., in concert with oligonucleotide synthesis/ gene reassembly techniques. Any of the preceding general recombination formats can be practiced in a reiterative fashion to generate a more diverse set of recombinant nucleic acids.
• nucleic acids of the invention can be recombined (with each other, or with related (or even unrelated) nucleic acids to produce a diverse set of recombinant nucleic acids, including e.g., homologous nucleic acids.
• sequence recombination techniques described herein provide particular advantages in that they provide for recombination between the nucleic acids of SEQ ID NO:l to SEQ ID NO:35, and SEQ ID NO:72 to SEQ ID NO:78, or fragments or variants thereof, in any available format, thereby providing a very fast way of exploring the manner in which different combinations of sequences can affect a desired result.
• any nucleic acids which are produced can be screened or selected for a desired activity.
• this can include testing for and identifying any activity that can be detected, e.g., in an automatable format, by any assay known in the art.
• useful properties such as low immunogenicity, increased half-life, improved solubility, oral availability, or the like can also be selected for.
• a variety of alpha-interferon related (or even unrelated) properties can be assayed for, using any available assay.
• DNA mutagenesis and shuffling provide a robust, widely applicable, means of generating diversity useful for the engineering of proteins, pathways, cells and organisms with improved characteristics.
• Mutagenesis methods of generating diversity include, for example, recombination (PCT/US98/05223; Publ. No. W098/42727); site-directed mutagenesis (Ling et al. (1997) “Approaches to DNA mutagenesis: an overview," Anal. Biochem. 254(2): 157-178; Dale et al. (1996) “Oligonucleotide-directed random mutagenesis using the phosphorothioate method," Methods Mol. Biol. 57:369-374; Smith (1985) "In vitro mutagenesis," Ann. Rev. Genet.
• Random or semi-random mutagenesis using doped or degenerate oligonucleotides (Arkin and Youvan (1992) "Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis," Biotechnology 10:297- 300; Reidhaar-Olson et al. (1991) "Random mutagenesis of protein sequences using oligonucleotide cassettes," Methods Enzymol. 208:564-86; Lim and Sauer (1991) "The role of internal packing interactions in determining the structure and stability of a protein," J. Mol. Biol.
• enor-prone PCR can be used to generate nucleic acid variants. Using this technique, PCR is performed under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product.
• assembly PCR can be used, in a process which involves the assembly of a PCR product from a mixture of small DNA fragments.
• a large number of different PCR reactions can occur in parallel in the same vial, with the products of one reaction priming the products of another reaction.
• Sexual PCR mutagenesis can be used in which homologous recombination occurs between DNA molecules of different but related DNA sequence in vitro, by random fragmentation of the DNA molecule based on sequence homology, followed by fixation of the crossover by primer extension in a PCR reaction.
• Recursive ensemble mutagenesis can be used in which an algorithm for protein mutagenesis is used to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Examples of this approach are found in Arkin & Youvan (1992) Proc. Nat'l Acad. Sci. USA 89:7811-7815.
• oligonucleotide directed mutagenesis can be used in a process which allows for the generation of site-specific mutations in any nucleic acid sequence of interest.
• cassette mutagenesis can be used in a process which replaces a small region of a double stranded DNA molecule with a synthetic oligonucleotide cassette that differs from the native sequence.
• the oligonucleotide can contain, e.g., completely and/or partially randomized native sequence(s).
• In vivo (or ex vivo) mutagenesis can be used in a process of generating random mutations in any cloned DNA of interest which involves the propagation of the DNA, e.g., in a strain of E. coli that carries mutations in one or more of the DNA repair pathways. These "mutator" strains have a higher random mutation rate than that of a wild- type parent. Propagating the DNA in one of these strains will eventually generate random mutations within the DNA.
• Exponential ensemble mutagenesis can be used for generating combinatorial libraries with a high percentage of unique and functional mutants, where small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins.
• Kits for mutagenesis, library construction, and other diversity generation methods are also commercially available.
• kits are available from, e.g., Stratagene (e.g., QuickChangeTM site-directed mutagenesis kit; and ChameleonTM double- stranded, site-directed mutagenesis kit), Bio/Can Scientific, Bio-Rad (e.g., using the
• chimeric nucleic acid multimers suitable for transformation into a variety of species
• techniques have been proposed which produce chimeric nucleic acid multimers suitable for transformation into a variety of species (see, e.g., Schellenberger U.S. Patent No. 5,756,316 and the references above).
• chimeric multimers consist of genes that are divergent with respect to one another (e.g., derived from natural diversity or through application of site directed mutagenesis, enor prone PCR, passage through mutagenic bacterial strains, and the like), are transformed into a suitable host, this provides a source of nucleic acid diversity for DNA diversification.
• Chimeric multimers transformed into host species are suitable as substrates for in vivo (or ex vivo) shuffling protocols.
• a multiplicity of polynucleotides sharing regions of partial sequence similarity or homology can be transformed into a host species and recombined in vivo (or ex vivo) by the host cell. Subsequent rounds of cell division can be used to generate libraries, members of which, comprise a single, homogenous population of monomeric or pooled nucleic acid. Alternatively, the monomeric nucleic acid can be recovered by standard techniques and recursively recombined in any of the described shuffling formats.
• the single stranded polynucleotides are then annealed and incubated in the presence of a polymerase and a chain terminating reagent (e.g., ultraviolet, gamma or X- ray inadiation; ethidium bromide or other intercalators; DNA binding proteins, such as single strand binding proteins, transcription activating factors, or histones; polycyclic aromatic hydrocarbons; trivalent chromium or a trivalent chromium salt; or abbreviated polymerization mediated by rapid thermocycling; and the like), resulting in the production of partial duplex molecules.
• a chain terminating reagent e.g., ultraviolet, gamma or X- ray inadiation; ethidium bromide or other intercalators; DNA binding proteins, such as single strand binding proteins, transcription activating factors, or histones; polycyclic aromatic hydrocarbons; trivalent chromium or a trivalent chromium salt; or abbreviated polymerization
• the partial duplex molecules e.g., containing partially extended chains, are then denatured and reannealed in subsequent rounds of replication or partial replication resulting in polynucleotides which share varying degrees of sequence similarity or homology and which are chimeric with respect to the starting population of DNA molecules.
• the products or partial pools of the products can be amplified at one or more stages in the process.
• Polynucleotides produced by a chain termination method, such as described above are suitable substrates for diversity generation methods (e.g., RSR, DNA shuffling) according to any of the described formats.
• Diversity can be further increased by using methods which are not homology based with DNA shuffling (which, as set forth in the above publications and applications can be homology or non-homology based, depending on the precise format).
• incremental truncation for the creation of hybrid enzymes (ITCHY) described in Ostermeier et al. (1999) "A combinatorial approach to hybrid enzymes independent of DNA homology” Nature Biotech. 17:1205 can be used to generate an initial recombinant library which serves as a substrate for one or more rounds of in vitro, ex vivo, or in vivo diversity generation methods (e.g., RSR or shuffling methods).
• Methods for generating multispecies expression libraries have been described (e.g., U.S. Patent Nos.
• Multispecies expression libraries are, in general, libraries comprising cDNA or genomic sequences from a plurality of species or strains, operably linked to appropriate regulatory sequences, in an expression cassette.
• the cDNA and/or genomic sequences are optionally randomly concatenated to further enhance diversity.
• the vector can be a shuttle vector suitable for transformation and expression in more than one species of host organism, e.g., bacterial species, eukaryotic cells.
• the library is biased by preselecting sequences which encode a protein of interest, or which hybridize to a nucleic acid of interest. Any such libraries can be provided as substrates for any of the methods herein described.
• preselect or prescreen libraries e.g., an amplified library, a genomic library, a cDNA library, a normalized library, etc.
• substrate nucleic acids e.g., an amplified library, a genomic library, a cDNA library, a normalized library, etc.
• shuffling procedures can also, independently have these effects.
• recombined CDRs derived from B cell cDNA libraries can be amplified and assembled into framework regions (e.g., Jirholt et al. (1998) "Exploiting sequence space: shuffling in vivo formed complementarity determining regions into a master framework," Gene 215:471) prior to diversity generation (e.g., DNA shuffling) according to any of the methods described herein.
• Libraries can be biased towards nucleic acids which encode proteins with desirable activities (e.g., binding affinities, enzymatic activities, anti-viral activities, ability to induce an immune response, antiproliferative activities, adjuvant properties, etc.).
• the clone can be mutagenized using any known method for introducing DNA alterations, including, but not restricted to, DNA shuffling or another form of recursive sequence recombination or diversity generation.
• a library comprising the mutagenized homologues is then screened for a desired activity, which can be the same as or different from the initially specified activity.
• Desired activities can be identified by any method known in the art.
• WO 99/10539 proposes that gene libraries can be screened by combining extracts from the gene library with components obtained from metabolically rich cells and identifying combinations which exhibit the desired activity.
• clones with desired activities can be identified by inserting bioactive substrates into samples of the library, and detecting bioactive fluorescence conesponding to the product of a desired activity using a fluorescent analyzer, e.g., a flow cytometry device, a CCD, a fluorometer, or a spectrophotometer.
• a fluorescent analyzer e.g., a flow cytometry device, a CCD, a fluorometer, or a spectrophotometer.
• Libraries can also be biased towards nucleic acids which have specified characteristics, e.g., hybridization to a selected nucleic acid probe.
• a desired activity e.g., an enzymatic activity, for example: a lipase, an esterase, a protease, a glycosidase, a glycosyl transferase, a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, a hydratase, a nitrilase, a transaminase, an amidase or an acylase) can be identified from among genomic DNA sequences in the following manner.
• an enzymatic activity for example: a lipase, an esterase, a protease, a glycosidase, a glycosyl transferase, a phosphatase, a kinase, an oxygenase, a peroxidase
• genomic DNA Single stranded DNA molecules from a population of genomic DNA are hybridized to a ligand-conjugated probe.
• the genomic DNA can be derived from either a cultivated or uncultivated microorganism, or from an environmental sample. Alternatively, the genomic DNA can be derived from a multicellular organism, or a tissue derived therefrom.
• Second strand synthesis can be conducted directly from the hybridization probe used in the capture, with or without prior release from the capture medium or by a wide variety of other strategies known in the art.
• the isolated single- stranded genomic DNA population can be fragmented without further cloning and used directly in a shuffling-based gene reassembly process.
• the fragment population derived the genomic library(ies) is annealed with partial, or, often approximately full length ssDNA or RNA conesponding to the opposite strand. Assembly of complex chimeric genes from this population is the mediated by nuclease-base removal of non-hybridizing fragment ends, polymerization to fill gaps between such fragments and subsequent single stranded ligation.
• the parental strand can be removed by digestion (if RNA or uracil-containing), magnetic separation under denaturing conditions (if labeled in a manner conducive to such separation) and other available separation/purification methods.
• the parental strand is optionally co-purified with the chimeric strands and removed during subsequent screening and processing steps.
• Non-Stochastic methods of generating nucleic acids and polypeptides are alleged in Short, J. "Non-Stochastic Generation of Genetic Vaccines and Enzymes," WO 00/46344. These methods, including the proposed non-stochastic polynucleotide reassembly and gene site saturation mutagenesis and synthetic ligation polynucleotide reassembly methods outlined therein, can be applied to the present invention as well. It will readily be appreciated that any of the above described techniques suitable for enriching a library prior to diversification can also be used to screen the products, or libraries of products, produced by the diversity generating methods.
• a recombinant nucleic acid produced by recursively recombining one or more polynucleotides of the invention with one or more additional nucleic acids also forms a part of the invention.
• the one or more additional nucleic acids may include another polynucleotide of the invention; optionally, alternatively, or in addition, the one or more additional nucleic acids can include, e.g., a nucleic acid encoding a naturally- occurring interferon-alpha or a subsequence thereof, or any homologous interferon-alpha sequence or subsequence thereof, or an interferon-beta sequence or subsequence thereof (e.g., an interferon-alpha or interferon-beta sequence as found in GenBank or other available literature), or, e.g., any other homologous or non-homologous nucleic acid
• the recombining steps may be performed in vivo, ex vivo, in vitro, or in silico as described in more detail in the references above.
• a cell containing any resulting recombinant nucleic acid, nucleic acid libraries produced by diversity generation, recombination, or recursive recombination of the nucleic acids set forth herein, and populations of cells, vectors, viruses, plasmids or the like comprising the library or comprising any recombinant nucleic acid resulting from diversity generation or recombination (or recursive recombination) of a nucleic acid as set forth herein with another such nucleic acid, or an additional nucleic acid.
• Conesponding sequence strings in a database present in a computer system or computer readable medium are a feature of the invention.
• the invention also includes compositions comprising two or more polynucleotides of the invention (e.g., as substrates for recombination).
• the composition can comprise a library of recombinant nucleic acids, where the library contains at least 2, 3, 5, 10, 20, or 50 or more nucleic acids.
• the nucleic acids are optionally cloned into expression vectors, providing expression libraries.
• the invention also includes compositions produced by digesting one or more polynucleotides of the invention with a restriction endonuclease, an RNAse, or a DNAse (e.g., as is performed in certain of the recombination formats noted above); and compositions produced by fragmenting or shearing one or more polynucleotides of the invention by mechanical means (e.g., sonication, vortexing, and the like), which can also be used to provide substrates for recombination in the methods above.
• compositions comprising sets of oligonucleotides conesponding to more than one nucleic acids of the invention are useful as recombination substrates and are a feature of the invention. For convenience, these fragmented, sheared, or oligonucleotide synthesized mixtures are refened to as fragmented nucleic acid sets.
• compositions produced by incubating one or more of the fragmented nucleic acid sets in the presence of ribonucleotide- or deoxyribonucelotide triphosphates and a nucleic acid polymerase are also included in the invention.
• the nucleic acid polymerase may be an RNA polymerase, a DNA polymerase, or an RNA-directed DNA polymerase (e.g., a "reverse transcriptase"); the polymerase can be, e.g., a thermostable DNA polymerase (such as, VENT, TAQ, or the like).
• the invention provides isolated or recombinant interferon-alpha homologue polypeptides, also refened to herein as "interferon-alpha homologues," or “interferon homologues” or “IFN-alpha homologues” or “EFN homologues”.
• An isolated or recombinant interferon homologue polypeptide of the invention includes a polypeptide comprising a sequence selected from SEQ ID NO:36 to SEQ ED NO:70 and SEQ ID
• the invention also provides a polypeptide comprising at least about 100, 120, 130, 140, 150, 155, 160, 163, 165, or 166 contiguous amino acids of any one of SQ ID NOS:36-70 or SEQ ID NO:71.
• said amino acid sequence comprises amino acids Lysl60 and Glul66, wherein the numbering of the amino acids in the sequence conesponds to that of SEQ ID NO:36.
• interferon homologue polypeptide sequences of the invention of the following amino acid residues (denoted "Group I” residues) which do not appear in the equivalent position of known, naturally- occurring human or non-human Type 1 interferon sequences.
• interferon homologue polypeptide sequences of the invention of the following amino acid residues (denoted "Group II” residues) which do not appear in the equivalent position of known, naturally-occurring human interferon-alpha subtype sequences.
• Group II Pro9; (Lys, Ser)12; (Thr, Val)24; Gln34; Arg40; Ser45; Arg47;
• an interferon polypeptide comprises an amino acid sequence comprising a proline residue at amino acid position 9 in the sequence, a lysine or serine residue at position 12, a threonine or valine residue at position 24, a glutamine residue at position 34, an arginine residue at position 40, etc.
• Such polypeptides may exhibit antiproliferative activities in a human Daudi cell line-based proliferation assay (e.g., at least about 8.3xl0 6 units/mg) and/or an antiviral activities in a human WISH cell/EMCV-based assay (at least about 2.1xl0 7 units/mg).
• Some such polypeptides bind a human alpha interferon receptor.
• polypeptides are 166 amino acids in length.
• polypeptides may comprise a sequence selected from any of the group of SEQ ID NO:36 to SEQ ID NO:54.
• An antiproliferative activity of any polypeptide of the invention generally relates to the capability or ability of a polypeptide to cause cells or parts thereof to grow or produce new cellular growth rapidly and often repeatedly.
• the invention further includes a polypeptide (e.g., any of SEQ ID NOS:36- 71 or SEQ ID NOS:79-85) or a nucleic acid (e.g., any of SEQ ID NOS:l-35 or SEQ ID NOS:72-78)encoding a polypeptide, wherein said polypeptide having an anti-angiogenic activity as measured by an anti-angiogenesis assay well known to those of ordinary skill in the art.
• a polypeptide e.g., any of SEQ ID NOS:36- 71 or SEQ ID NOS:79-85
• a nucleic acid e.g., any of SEQ ID NOS:l-35 or SEQ ID NOS:72-78
• the invention further includes:
• any interferon-alpha polypeptide comprising one or more Group II amino acid residues above in the context of a human like interferon sequence (i.e., a sequence which displays a high level of similarity or homology to a human interferon), or a sequence which is highly similar or homologous (i.e., having a percent sequence homology or sequence identity of at least about 80%, 90%, 95%, 96%, 97%, 98% or more) to any sequence listed in the attached sequence listing or fragment thereof.
• a human like interferon sequence i.e., a sequence which displays a high level of similarity or homology to a human interferon
• a sequence which is highly similar or homologous i.e., having a percent sequence homology or sequence identity of at least about 80%, 90%, 95%, 96%, 97%, 98% or more
• the present invention provides an interferon alpha homologue comprising the sequence show in SEQ ID NO:71: CDLPQTHSLG-X ⁇ -X] 2 - RA-X 15 -X 16 -LL-X 19 -QM-X 22 -R-X 24 -S-X 26 -FSCLKDR-X 34 -DFG-X 38 -P-X 4 o-EEFD-X 45 - X 46 -X 47 -FQ-X 5 o-X 51 -QAI-X 55 -X 56 -X 57 -HE-X 60 -X 61 -QQTFN-X 67 -FSTK-X 72 -SS-X 75 -X 76 - W-X 78 -X 79 -X 80 -LL-X 83 -K-X 85 -X 86 -T-X 88 -L-X 90 -QQLN-X 95 -LEACV-X 1 o ⁇ -Q-X
• the interferon homologue polypeptide of SEQ ID NO:71 exhibits an antiproliferative activity in a human Daudi cell line-based proliferation assay (at least about 8.3xl0 6 units/mg) and/or an antiviral activity in a human WISH cell/EMCV-based assay (at least about 2.1xl0 7 units/mg). Both such assays are discussed in greater detail below.
• Such polypeptide may comprise an amino acid sequence of the group of from SEQ ED NO:36 to SEQ ED NO:54 or may be encoded by a nucleotide sequence of the group of from SEQ ID NO:l to SEQ ID NO: 19.
• An interferon alpha homologue fragment of the invention typically comprises an interferon homologue polypeptide comprising at least about 20, 25, or 30, and typically at least about 40, 50, 60, 70, 80, 90, or 100 contiguous amino acids of any one of SEQ ID NOS:36-71 or SEQ ID NOS:79-85.
• the fragment comprises usually at least about 100, 110, 120, 125, 130, 140, 150, 155, 158, 160, 162, 163, 164, or 165 contiguous amino acids of any one of SEQ ID NOS:36-71 or SEQ ID NOS:79-85.
• Such polypeptide fragments may have an antiproliferative activity in a human Daudi cell line-based assay and/or an antiviral activity in a human or murine cell line/EMCV-based assay.
• the invention provides polypeptides having a length of 166 amino acids, and, in some such embodiments, such polypeptides have an antiproliferative activity in a human Daudi cell line-based assay (or other similar assay), including, e.g., at least about 8.3xl0 6 units/mg, and/or an antiviral activity in a human WISH cell line/EMCV-based assay (or other similar assay), including, e.g., at least about 2.1x10 units/mg.
• the invention provides a polypeptide comprising at least 100, 150, 155, or 160 contiguous amino acids of a protein encoded by a coding polynucleotide sequence comprising any of the following: (a) SEQ ID NO: 1 to SEQ ID NO:35 or SEQ ID NO:72 to SEQ ED NO:78; (b) a coding polynucleotide sequence that encodes a first polypeptide selected from any of SEQ ID NO:36 to SEQ ID NO:70 or SEQ ED NO:79 to SEQ ED NO:85; and (c) a complementary polynucleotide sequence that hybridizes under at least highly stringent (or ultra-high stringent or ultra-ultra- high stringent conditions) hybridization conditions over substantially the entire length of a polynucleotide sequence of (a) or (b).
• Such polypeptides may have an antiproliferative activity in a human Daudi cell line-based assay (or other similar assay), and/or an antiviral activity in a human WISH cell line/EMCV-based assay (or other similar assay).
• Some such polypeptides of the invention specifically bind a human alpha interferon receptor.
• polypeptides and nucleic acids of the subject invention need not be identical, but can be substantially identical, to the conesponding sequence of the target molecule or related molecule, including the polypeptides of any of SEQ ID NOS:36-71 or fragments thereof (including those having antiviral or antiproliferative activities in the assays described herein), or the nucleic acids of any of SEQ ID NOS: 1-35 or fragments thereof (including those having antiviral or antiproliferative activities in the assays described herein).
• the polypeptides can be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use.
• the polypeptides of the invention can be modified in a number of ways so long as they comprise a sequence substantially identical (as defined below) or having a percent identity to a sequence in the naturally occurring or known interferon polypeptide molecule.
• sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over a window of comparison.
• percentage of sequence identity or “percent sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• the present invention provides interferon homologue nucleic acids having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more percent sequence identity with the nucleic acids of any of SEQ ID NOS: 1-35 or SEQ ID NOS:72-78 or fragments thereof.
• substantial identity means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights (described in detail below), share at least about 80 percent sequence identity, preferably at least about 90 percent sequence identity, more preferably at least about 95 percent sequence identity or more (e.g., 97, 98, or 99 percent sequence identity).
• residue positions which are not identical differ by conservative amino acid substitutions.
• Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
• a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
• a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
• a group of amino acids having amide-containing side chains is asparagine and glutamine
• a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
• a group of amino acids having basic side chains is lysine, arginine, and histidine
• a group of amino acids having sulfur-containing side chains is cysteine and methionine.
• Prefened conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine- glutamine.
• the present invention provides interferon homologue polypeptides having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% 99% 99.5% or more percent sequence identity with the polypeptides of any of SEQ ED NOS:36-71 or SEQ ID NOS :79-85 or fragments thereof.
• BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: //www.ncbi. nlm.nih.gov/).
• This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is refened to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
• a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) Proc. Nat'l Acad. Sci. U.S.A. 90: 5873-5787).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
• PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment.
• PILEUP uses a simplification of the progressive alignment method of Feng & Doolittie (1987) J. Mol. Evol. 35: 351-360. The method used is similar to the method described by Higgins & Sha ⁇ (1989) CABIOS 5: 151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
• the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
• the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
• PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
• PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al. (1984) Nuc. Acids Res. 12: 387-395.
• ClustalW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties were 10 and 0.05, respectively.
• BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff (1992) Proc. Nat'lAcad. Sci. U.S.A. 89: 10915- 10919).
• polypeptides of the invention Recombinant methods for producing and isolating interferon homologue polypeptides of the invention are described above.
• the polypeptides may be produced by direct peptide synthesis using solid-phase techniques (cf. Stewart et al. (1969) Solid-Phase Peptide Synthesis, W.H. Freeman Co, San Francisco; Merrifield, J. (1963) J. Am. Chem. Soc. 85:2149-2154). Peptide synthesis may be performed using manual techniques or by automation.
• Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with the instructions provided by the manufacturer.
• subsequences may be chemically synthesized separately and combined using chemical methods to provide full-length interferon homologues.
• Fragments of the interferon homologue polypeptides of the invention are also a feature of the invention and may be synthesized by using the procedures described above.
• Polypeptides of the invention can be produced by introducing into a population of cells a nucleic acid of the invention, wherein the nucleic acid is operatively linked to a regulatory sequence effective to produce the encoded polypeptide, culturing the cells in a culture medium to produce the polypeptide, and optionally isolating the polypeptide from the cells or from the culture medium.
• polypeptides of the invention can be produced by introducing into a population of cells a recombinant expression vector comprising at least one nucleic acid of the invention, wherein the at least one nucleic acid is operatively linked to a regulatory sequence effective to produce the encoded polypeptide, culturing the cells in a culture medium under suitable conditions to produce the polypeptide encoded by the expression vector, and optionally isolating the polypeptide from the cells or from the culture medium.
• an interferon homologue polypeptide of the invention is used to produce antibodies which have, e.g., diagnostic, prophylactic and therapeutic uses, e.g., related to the activity, distribution, and expression of interferon homologues.
• Antibodies to interferon homologues of the invention may be generated by methods well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Antibodies, i.e., those which block receptor binding, are especially prefened for therapeutic or prophylactic use.
• Interferon homologue polypeptides for antibody induction do not require biological activity; however, the polypeptide or oligopeptide must be antigenic.
• Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least 10 amino acids, preferably at least 15 or 20 amino acids. Short stretches of an interferon homologue polypeptide may be fused with another protein, such as keyhole limpet hemocyanin, and antibody produced against the chimeric molecule.
• Suitable techniques for antibody preparation include selection of libraries of recombinant antibodies in phage or similar vectors. See, Huse et al. (1989) Science 246:1275-1281; and Ward et al. (1989) Nature 341:544-546. Specific monoclonal and polyclonal antibodies and antisera will usually bind with a K D of at least about 0.1 ⁇ M, preferably at least about 0.01 ⁇ M or better, and most typically and preferably, 0.001 ⁇ M or better.
• this invention provides for fully humanized antibodies against the interferon homologues of the invention.
• Humanized antibodies are especially desirable in applications where the antibodies are used as prophylactics and therapeutics in vivo and ex vivo in human patients.
• Human antibodies consist of characteristically human immunoglobulin sequences.
• the human antibodies of this invention can be produced in using a wide variety of methods (see, e.g., Larrick et ai, U.S. Pat. No. 5,001,065, and Bonebaeck McCafferty and Paul, supra, for a review).
• the human antibodies of the present invention are produced initially in trioma cells.
• Triomas Genes encoding the antibodies are then cloned and expressed in other cells, such as nonhuman mammalian cells.
• the general approach for producing human antibodies by trioma technology is described by Ostberg et al. (1983), Hybridoma 2:361- 367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al, U.S. Pat. No. 4,634,666.
• the antibody-producing cell lines obtained by this method are called triomas because they are descended from three cells; two human and one mouse. Triomas have been found to produce antibody more stably than ordinary hybridomas made from human cells.
• the interferon homologue polypeptides of the present invention or fragments thereof are useful as adjuvants to stimulate, enhance, potentiate, or augment an immune response related to an antigen when administered together with the antigen or after or before delivery of the antigen.
• the invention provides methods for administering one or more of the polypeptides invention described herein to a subject.
• interferon homologue polypeptides of the present invention or fragments thereof are useful in the prophylactic and/or therapeutic treatment of a variety of diseases, disorders, or medical conditions.
• the invention provides interferon-alpha homologue polypeptides (and interferon-alpha homologue nucleic acids which encode such polypeptides) that have both antiviral and antiproliferative activities in the assays described herein.
• the invention provides interferon-alpha homologue polypeptides (and interferon-alpha homologue nucleic acids which encode such polypeptides) in which the ratio of antiviral activity to antiproliferative activity is greater than that of other known interferon-alphas such as those listed in GenBank as noted herein.
• Such polypeptides are useful in the therapeutic and/or prophylactic treatment of various diseases and disorders, such as, e.g., treatment regimens for hepatitis B, hepatitis C, HIV, and HSV.
• some such polypeptides (and nucleic acids encoding them) such as interferon-alpha homologue 2BA8, offer significant advantages over known interferon-alpha compounds, since they likely exhibit lower side effects upon administration than known interferon-alpha compounds, such as interferon-alpha 2a, are of higher potency, and thus may require in lower dosing and cause fewer immunogenicity effects.
• Interferon homologue polypeptides of the present invention include one or more conservatively modified variations (or “conservative variations” or conservative substitutions") of the polypeptide sequences disclosed herein as SEQ ID NO:36 to SEQ ID NO:70 and SEQ ID NO:79 to SEQ ID NO:85.
• conservatively modified variations comprise substitutions, additions or deletions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than about 5%, more typically less than about 4%, 2%, or 1%) in any of SEQ ED NO:36 to SEQ ID NO:70 and SEQ ID NO:79 to SEQ ID NO:85.
• a conservatively modified variation (e.g., deletion) of the 166 amino acid polypeptide identified herein as SEQ ED NO:36 has a length of at least about 157 or 158 amino acids, preferably at least about 159 or 160 amino acids, more preferably at least about 162 or 163 amino acids, and still more preferably at least about 164 or 165 amino acids, conesponding to a deletion of less than about 5%, 4%, 2% or 1% of the polypeptide sequence, respectively.
• a conservatively modified variation e.g., a "conservatively substituted variation” of the polypeptide identified herein as SEQ ID NO:36 will contain "conservative substitutions", according to the six substitution groups set forth in Table 2 (supra), in up to about 8 residues (i.e., less than about 5%) of the 166 amino acid polypeptide.
• interferon homologue polypeptide sequences of the invention can be present as part of larger polypeptide sequences such as which occur upon the addition of one or more domains for purification of the protein (e.g., poly His segments, FLAG epitope segments, etc.), e.g., where the additional functional domains have little or no effect on the activity of the interferon-alpha portion of the protein, or where the additional domains can be removed by post synthesis processing steps such as by treatment with a protease.
• domains for purification of the protein e.g., poly His segments, FLAG epitope segments, etc.
• interferon homologue polypeptides of the present invention comprise the following sequence, identified herein as SEQ ED NO:71: CDLPQTHSLG-X 1 ⁇ -Xi 2 -RA-X 15 -Xi 6 -LL-X ⁇ 9 -QM-X 22 -R-X 24 -S-X 26 -FSCLKDR-X 34 - DFG-X 38 -P-X 40 -EEFD-X 45 -X 46 -X 47 -FQ-X 5 o-X 5 i-QAI-X 55 -X 56 -X 57 -HE-X 60 -X 61 -QQTFN- X 67 -FSTK-X 72 -SS-X 75 -X 76 -W-X 78 -X 79 -X 80 -LL-X 83 -K-X 85 -X 86 -T-X 88 -L-X 90 -QQLN-X 95 - LEACV-X
• X H is N or D
• X 12 is R, S, or K
• X 15 is L or M
• X ⁇ 6 is I, M, or V
• X 19 is A or G
• X 2 is G or R
• X 24 is I or T
• X 26 is P or H
• X 3 is H, Y or Q
• X 38 is F or L
• X 40 is Q or R
• X 45 is G or S
• X 46 is N or H
• X 47 is Q or R
• X 50 is K or R
• X 5 ⁇ is A or T
• X 55 is S or F
• X 56 is V or A
• X 57 is L or F
• X 60 is M or I
• X 6 ⁇ is I or M
• X 67 is L or F
• X 7 is D or
• a conservatively modified variation of the sequence of SEQ ID NO:71 can include up to a total of about 8 amino acid deletions, insertions, or conservative substitutions in the 166 amino acid polypeptide, excluding the positions designated X in SEQ ID NO:71, which conespond to the amino acid explicitly defined.
• WEVVR AEIMR SFSFS TNLQK RLRRKE include: WEVVR SEJMR SFS YS TNLQR RLRRKD and
• a feature of the invention is an interferon homologue polypeptide comprising at least about 20, usually at least about 25, typically at least about 30, 40, 50, 60, 70, 80, 90, or 100 contiguous amino acids of any one of SEQ ED NOS:36-71 or SEQ ID NOS:79-85.
• the polypeptide typically comprises at least about 100, 110, 120, 125, 130, 140, 150, 155, 158, 160, 163, 164, or 165 contiguous amino acids of any one of SEQ ID NOS:36-70 or SEQ ID NOS:79-85.
• the interferon homologue polypeptide of the invention comprises an amino acid sequence comprising one or more of amino acid residues (Tyr or Gln)34, Gly37, Phe38, Lys71, Ala76, Tyr90, Ilel32, Argl34, Phel52, Lysl60, and Glul66, wherein the numbering of the amino acids conesponds to the numbering of amino acids in the amino acid sequence of SEQ ID NO:36.
• the interferon homologue polypeptide comprises an amino acid sequence comprising at least 150, 155, or 166 contiguous amino acid residues of any one of SEQ ID NOS:36-70, further comprising Lysl60 and Glul66, wherein the numbering of the amino acids conesponds to the numbering of amino acids in the amino acid sequence of SEQ ID NO:36.
• Some such polypeptides also exhibit an antiproliferative activity of at least about 8.3xl0 6 units/milligram in the human Daudi cell line - based assay, or an antiviral activity of at about least 2.1xl0 7 units/milligram (mg) in the human WISH cell/EMCV-based assay.
• polypeptides of the invention provide a variety of new polypeptide sequences as compared to other alpha interferon homologues, the polypeptides also provide a new structural features which can be recognized, e.g., in immunological assays.
• the generation of antisera which specifically binds the polypeptides of the invention, as well as the polypeptides which are bound by such antisera, are features of the invention.
• the invention includes interferon-alpha homologue polypeptides that specifically bind to or that are specifically immunoreactive with an antibody or antisera generated against an immunogen comprising an amino acid sequence selected from one or more of SEQ ID NO:36 to SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:79 to SEQ ED NO:85.
• an antibody or antisera generated against an immunogen comprising an amino acid sequence selected from one or more of SEQ ID NO:36 to SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:79 to SEQ ED NO:85.
• the antibody or antisera or antiserum
• available known alpha interferons such as those polypeptides encoded by nucleic acids represented by GenBank accession numbers J00210 (alpha-D), J00207 (Alpha-A),
• X02958 (Alpha-6), X02956 (Alpha-5), V00533 (alpha-H), V00542 (alpha-14), V00545 (IFN-1B), X03125 (alpha-8), X02957 (alpha-16), V00540 (alpha-21), X02955 (alpha-4b), V00532 (alpha-C), X02960 (alpha-7), X02961 (alpha-10 pseudogene), R0067 (Gx-1), 101614, 101787, 107821, M12350 (alpha-F), and M38289, V00549 (alpha-2a), and 108313 (alpha-Conl), or any other known interferon-alpha polypeptides (typically refened to as the "control alpha interferon polypeptides").
• accession number conesponds to a nucleic acid a polypeptide encoded by the nucleic acid is generated and used for antibody/antisera subtraction pu ⁇ oses.
• nucleic acid conesponds to a non- coding sequence e.g., a pseudo gene
• an amino acid which conesponds to the reading frame of the nucleic acid is generated (e.g., synthetically), or is minimally modified to include a start codon for recombinant production.
• the immunoassay uses a polyclonal antiserum which was raised against one or more polypeptides comprising one or more of the amino acid sequences conesponding to one or more of: SEQ ID NO:36 to SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:79 to SEQ ID NO:85, or a substantial subsequence thereof (i.e., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% or more of the full length sequence provided).
• the full set of potential polypeptide immunogens derived from one or more of SEQ ID NO: 36 to SEQ ID NO: 70, SEQ ID NO: 7, and SEQ ID NO:79 to SEQ ID NO:85 are collectively refened to below as "the immunogenic polypeptides.”
• the resulting antisera is optionally selected to have low cross-reactivity against the control alpha interferon polypeptides and/or other known interferon polypeptides and any such cross-reactivity is removed by immunoabso ⁇ tion with one or more of the control alpha interferon polypeptides, prior to use of the polyclonal antiserum in the immunoassay.
• one or more of the immunogenic polypeptides is produced and purified as described herein.
• recombinant protein may be produced in a mammalian cell line.
• An inbred strain of mice (used in this assay because results are more reproducible due to the virtual genetic identity of the mice) is immunized with the immunogenic polypeptide(s) in combination with a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
• one or more synthetic or recombinant polypeptides derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.
• Polyclonal sera are collected and titered against the immunogenic polypeptide(s) in an immunoassay, for example, a solid phase immunoassay with one or more of the immunogenic polypeptides immobilized on a solid support.
• Polyclonal antisera with a titer of 10 6 or greater are selected, pooled and subtracted with the control alpha interferon polypeptides to produce subtracted pooled titered polyclonal antisera.
• the subtracted pooled titered polyclonal antisera are tested for cross reactivity against the control alpha interferon polypeptides.
• Preferably at least two of the immunogenic alpha interferon polypeptides are used in this determination, preferably in conjunction with at least two of the control alpha interferon polypeptides, to identify antibodies which are specifically bound by the immunogenic polypeptides(s).
• discriminatory binding conditions are determined for the subtracted titered polyclonal antisera which result in at least about a 5-10 fold higher signal to noise ratio for binding of the titered polyclonal antisera to the immunogenic alpha interferons as compared to binding to the control alpha interferons . That is, the stringency of the binding reaction is adjusted by the addition of non-specific competitors such as albumin or non-fat dry milk, or by adjusting salt conditions, temperature, or the like. These binding conditions are used in subsequent assays for determining whether a test polypeptide is specifically bound by the pooled subtracted polyclonal antisera.
• test polypeptides which show at least a 2-5x higher signal to noise ratio than the control polypeptides under discriminatory binding conditions, and at least about a Vi signal to noise ratio as compared to the immunogenic polypeptide(s), shares substantial structural similarity or homology with the immunogenic polypeptide as compared to known alpha interferons, and is, therefore a polypeptide of the invention.
• immunoassays in the competitive binding format are used for detection of a test polypeptide.
• cross-reacting antibodies are removed from the pooled antisera mixture by immunoabso ⁇ tion with the control alpha interferon polypeptides.
• the immunogenic polypeptide(s) are then immobilized to a solid support which is exposed to the subtracted pooled antisera.
• Test proteins are added to the assay to compete for binding to the pooled subtracted antisera.
• test protein(s) The ability of the test protein(s) to compete for binding to the pooled subtracted antisera as compared to the immobilized protein(s) is compared to the ability of the immunogenic polypeptide(s) added to the assay to compete for binding (the immunogenic polypeptides compete effectively with the immobilized immunogenic polypeptides for binding to the pooled antisera).
• the percent cross-reactivity for the test proteins is calculated, using standard calculations.
• the ability of the control proteins to compete for binding to the pooled subtracted antisera is determined as compared to the ability of the immunogenic polypeptide(s) to compete for binding to the antisera. Again, the percent cross-reactivity for the control polypeptides is calculated, using standard calculations. Where the percent cross-reactivity is at least 5-10x as high for the test polypeptides, the test polypeptides are said to specifically bind the pooled subtracted antisera.
• the immunoabsorbed and pooled antisera can be used in a competitive binding immunoassay as described herein to compare any test polypeptide to the immunogenic polypeptide(s).
• the two polypeptides are each assayed at a wide range of concentrations and the amount of each polypeptide required to inhibit 50% of the binding of the subtracted antisera to the immobilized protein is determined using standard techniques. If the amount of the test polypeptide required is less than twice the amount of the immunogenic polypeptide that is required, then the test polypeptide is said to specifically bind to an antibody generated to the immunogenic polypeptide, provided the amount is at least about 5-10x as high as for a control polypeptide.
• the pooled antisera is optionally fully immunosorbed with the immunogenic polypeptide(s) (rather than the control polypeptides) until little or no binding of the resulting immunogenic polypeptide subtracted pooled antisera to the immunogenic polypeptide(s) used in the immunoabso ⁇ tion is detectable.
• This fully immunosorbed antisera is then tested for reactivity with the test polypeptide. If little or no reactivity is observed (i.e., no more than 2x the signal to noise ratio observed for binding of the fully immunosorbed antisera to the immunogenic polypeptide), then the test polypeptide is specifically bound by the antisera elicited by the immunogenic protein.
• Fig. 2 shows the antiproliferative activity of exemplary interferon homologues of the invention comprising amino acid sequences SEQ ID NO:36 to SEQ ID NO:54, in comparison to control interferons, human IFN-alpha 2a and consensus human IFN-alpha (Conl).
• the graph shows the number of Units of activity per milligram (mg) of interferon test sample (Y axis) for a set of exemplary interferon alpha homologues, each of which is designated with a name (clone name) on the X axis, compared with that of human IFN-alpha 2a and consensus human IFN-alpha.
• Interferon-alpha homologues of the present invention show diverse activity patterns against a variety of cancer cell lines (see, e.g., Example 2).
• An in vitro cell line screen (as described in, e.g., Monks, A. et al. (1991) J. Nat'l Cancer Inst. 83:757-766) was used to assay interferon-alpha homologues of the invention for selective growth inhibition and/or cell killing of particular cancer cell lines.
• the human cancer cell lines screened include leukemias, melanomas, and cancers of the lung, colon, brain, central nervous system, ovary, breast, prostate, and kidney.
• GI50 growth inhibition at 50%
• TGI total growth inhibition
• LC50 a measure of cytotoxic activity
• exemplary interferon-alpha homologue 3DA11 (SEQ ID NO:40) against a variety of cancer cell lines are shown in Figs. 3A, 3B, and 3C, in comparison with the interferon-alpha Conl and human interferon-alpha 2a controls.
• homologue 3DA11 and control interferon-alpha Conl showed significant activity against most of the cell lines tested, with the interferon-alpha Conl exhibiting generally higher activity, and interferon- alpha 2a generally exhibiting lower overall activity and in only a subset of the cell lines (Fig. 3A).
• homologue 3DA11 showed significant cytostatic activity against a population of cells of eleven of the cell lines, while interferon-Conl showed activity against only a population of cells of one of the cell lines, against which homologue 3DA11 was also active (Fig. 3B). IFN-alpha 2a, on the other hand, was not active in this assay against any of the tested cell lines.
• Homologue 3DA11 thus has a broader cytostatic activity profile than consensus human interferon-alpha (Conl) and human interferon-alpha 2a.
• Homologue 3DA11 also showed significant cytotoxic activity in comparison to the interferon-Conl and human interferon-alpha 2a controls (Fig. 3C).
• homologue 3DA11 displayed cytotoxic activity against a population of cells of 8 of the cell lines, whereas neither the interferon-Conl nor the interferon-alpha 2a controls exhibited measurable activity against a population of cells of any of the cell lines at the concentration range employed in the assay.
• homologue 3DA11 also has a broader cytotoxic activity profile than interferon-Conl and human interferon-alpha 2a.
• Figs. 4A-4D illustrate the cytostatic activity (as reflected by the TGI value) of exemplary interferon-alpha homologues of the invention.
• the relative cytostatic activity (expressed as -log TGI) against a population of cells of particular cancer cell line is plotted for various interferon-alpha homologues and for the two control interferons (interferon-Conl and human interferon-alpha 2a).
• the 1D3 and 3DA11 homologues showed at least about 25-fold higher cytostatic activity against a population of the cells (conesponding to a difference in TGI of at least about 1.4 log units) than did either of the controls (interferon-Conl or interferon-alpha 2a) against a population of cells of the leukemia cell line.
• Homologues 1D3, 2G5 (SEQ ID NO:45), 6CG3 (SEQ ID NO:52) and 3DA11 exhibited significant cytostatic activity against lung cancer cell line NCI-H23 (Fig. 4B).
• the 1D3, 2G5, 6CG3, and 3DA11 homologues showed at least about 12-fold higher cytostatic activity a population of cells of a lung cancer cell line (conesponding to a difference in TGI of at least about 1.1 log units) than either interferon-Conl or interferon-alpha 2a against a population of cells of the lung cancer cell line.
• Homologues 1D3, 2G5, and 3DA11 showed significant cytostatic activity against a population of cells of renal cancer cell line ACHN (Fig. 4C).
• the 1D3, 2G5, and 3DA11 homologues showed at least about 35-fold higher cytostatic activity a population of cells of said renal cancer cell line (conesponding to a difference in TGI of at least about 1.55 log units) than either interferon-Conl or interferon-alpha 2a against a population of cells of renal cancer cell line.
• homologue 1D3 showed at least about 2- fold higher cytostatic activity (conesponding to a difference in TGI of at least about 0.3 log units) than interferon-Conl
• the 1D3, 2G5, 3DA11, 2CA5, and 2DB11 homologues showed at least about 40-fold higher cytostatic activity (conesponding to a difference in TGI of at least about 1.6 log units) than interferon-alpha 2a, against respective populations of cells of the ovarian cancer cell line.
• interferon- alpha homologues of the invention showed a variety of cytostatic activity profiles, which differed significantly from those of the interferon-alpha Conl and interferon alpha-2a.
• the present invention includes an interferon-alpha homologue having increased cytostatic activity relative to human interferon-alpha 2a or to consensus human interferon-alpha, Conl.
• the interferon-alpha homologue has at least about 2-fold higher cytostatic activity a population of cells of a cancer cell line (i.e., has a TGI value at least about 2-fold lower) than does human interferon-alpha 2a, or has at least 2-fold higher cytostatic activity than interferon-Conl, against a population of cells of one or more cancer cell lines selected from the following: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a central nervous system (CNS) cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
• a leukemia cell line a melanoma cell line
• a lung cancer cell line a colon cancer cell line
• a central nervous system (CNS) cancer cell line an ovarian cancer cell line
• a breast cancer cell line a prostate cancer cell line
• a renal cancer cell line selected from the following:
• the interferon-alpha homologue has at least about 5- fold higher cytostatic activity a population of cells of a cancer cell line (i.e., has a TGI value at least about 5-fold lower) than does human interferon-alpha 2a, or has at least about 5-fold higher cytostatic activity than interferon-Conl, against a population of cells of one or more cancer cell lines selected from the following: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a central nervous system (CNS) cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
• a leukemia cell line a melanoma cell line
• a lung cancer cell line a colon cancer cell line
• a central nervous system (CNS) cancer cell line an ovarian cancer cell line
• a breast cancer cell line a prostate cancer cell line
• a renal cancer cell line selected from the following
• the interferon-alpha homologue has at least about 10-fold higher cytostatic activity a population of cells of a cancer cell line (i.e., has a TGI value at least about 10-fold lower) than does human interferon-alpha 2a, or has at least about 10-fold higher cytostatic activity than interferon-Conl, against a population of cells of one or more cancer cell lines selected from the following: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
• the invention includes an interferon-alpha homologue having increased cytotoxic activity relative to human interferon-alpha 2a or relative to interferon-Conl.
• the interferon-alpha homologue has at least about 2-fold higher cytotoxic activity (i.e., has an LC50 value at least about 2-fold lower), at least 5-fold higher cytotoxic activity, or at least 10-fold higher cytotoxic activity, than human interferon-alpha 2a against a population of cells of one or more cancer cell lines selected from the following: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
• the interferon-alpha homologue has at least about 2-fold higher cytotoxic activity (i.e., has an LC50 value at least about 2-fold lower), at least about 5-fold higher cytotoxic activity, or at least about 10-fold higher cytotoxic activity, than interferon-Conl, against a population of cells of at least one cancer cell line selected from: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
• a cancer cell line selected from: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
• the invention includes an interferon-alpha homologue having increased growth inhibition activity relative to human interferon-alpha 2a or to interferon-Conl.
• the interferon-alpha homologue has at least about 2-fold higher growth inhibition activity (i.e., has a GI50 value at least about 2-fold lower), at least about 5-fold higher growth inhibition activity, or at least about 10-fold higher growth inhibition activity, than human interferon-alpha 2a, against a population of cells of one or more cancer cell lines selected from: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
• the interferon-alpha homologue has at least about 2-fold higher growth inhibition activity (i.e., has a GI50 value at least about 2-fold lower), at least about 5-fold higher growth inhibition activity, or at least about 10-fold higher growth inhibition activity, than interferon-Conl, against at least one cancer cell line selected from the following: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
• a cancer cell line selected from the following: a leukemia cell line; a melanoma cell line; a lung cancer cell line; a colon cancer cell line; a CNS cancer cell line; an ovarian cancer cell line; a breast cancer cell line; a prostate cancer cell line; and a renal cancer cell line.
• interferons such as the interferon-alpha homologues described herein
• interferon-alpha homologues can be evolved, modified, or recombined to display a variety of activity profiles provides an opportunity for evolving and creating customized and specific interferon homologues for the treatment of a variety of specific diseases or disease conditions, including, e.g., a variety of cancers or related conditions.
• an interferon homologue of the invention optimized to have increased potency against a particular target cancer cell type may also be optimized to have (advantageously) reduced toxicity towards a non-target cell(s), and thus may produce lower side effects in the subject to which the homologue is administered (e.g., patient).
• the present invention further provides an opportunity to optimize interferon homologues against tumor cells taken from a subpopulation of subjects (e.g., mammals or human patients), or even from an individual subject (e.g., mammal or human patient), providing therapeutic or prophylactic treatment tailored to the individual subject.
• Optimized interferon homologues of the invention may provide therapeutic or prophylactic benefit against cancers or related conditions or other interferon-treatable disorders or conditions which are otherwise unresponsive to cunently-available interferons or to other treatment regimes.
• Fig. 2 shows the antiviral activity of exemplary interferon homologues of the invention comprising amino acid sequences SEQ ID NO:36 to SEQ ID NO:54.
• IFN-alpha homologues of the invention Improved in vitro antiviral activity of exemplary IFN-alpha homologues of the invention has been shown to be maintained in vivo in a murine model system.
• Two IFN-alpha homologues of the invention designated CH2.2 and CH2.3 (SEQ ID NOS:84 and 85, respectively), were previously shown to have about 206,000-fold and 138,000-fold improved antiviral activity, respectively, compared to human IFN-alpha 2a in a murine cell-based assay, as well as significantly higher activity in the same assay as compared to native murine interferons (Chang et al. (1999) Nature Biotechnol 17:793-797).
• VSV vesicular stomatitis virus
• compositions comprising interferon homologues of the present invention can be used in methods to inhibit viral replication in subjects infected with viruses including, but not limited to: human immunodeficiency virus (HIV), hepatitis C virus (HCV), he ⁇ es simplex virus (HSV), and hepatitis B virus (HBV).
• viruses including, but not limited to: human immunodeficiency virus (HIV), hepatitis C virus (HCV), he ⁇ es simplex virus (HSV), and hepatitis B virus (HBV).
• Inhibition can be performed in vitro ( useful, e.g., in a variety of antiviral assays), ex vivo (useful e.g., as a therapeutic or prophylactic agent in ex vivo methods discussed herein), or in vivo ( useful, e.g., as a therapeutic or prophylactic agent in in vivo methods discussed herein).
• compositions of the present invention can be used to therapeutically or prophylactically treat and thereby alleviate a variety of immune system-related disorders characterized by hyper- or hypo-active immune system function or other features.
• immune system-related disorders include hyperallergenicity and autoimmune disorders, such as multiple sclerosis, type I (insulin dependent) diabetes mellitus, lupus erythematosus, amyotrophic lateral sclerosis, Crohn's disease, rheumatoid arthritis, stomatitis, asthma, allergies, psoriasis and the like.
• compositions comprising one or more interferon homologue polypeptides or nucleic acids of the invention are tested in appropriate in vitro, ex vivo, and in vivo animal models of disease, to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art.
• dosages can be determined by activity comparison of the alpha interferon homologues to existing alpha interferon therapeutics or prophylactics, i.e., in a relevant assay.
• the invention provides methods comprising administering one or more interferon homologue nucleotides or polypeptides of the invention (or fragments thereof) described above to a mammal, including, e.g., a human, primate, mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, sheep; or a non-mammalian vertebrate such as a bird (e.g., a chicken or duck) or a fish, or invertebrate, as described in greater detail below.
• Such compositions typically comprise one or more interferon homologue nucleotides or polypeptides of the invention (or fragments thereof) and an excipient, including, e.g., a pharmaceutically acceptable excipient.
• composition of the invention is produced by digesting one or more nucleic acids of the invention (or fragments thereof) with a restriction endonuclease, an RNase, or a DNase.
• compositions produced by incubating one or more nucleic acids described above in the presence of deoxyribonucelotide triphosphates and a nucleic acid polymerase, e.g., a thermostable polymerase are provided.
• the invention also includes compositions comprising two or more nucleic acids described above.
• the composition may comprise a library of nucleic acids, where the library contains at least about 5, 10, 20, 50, 100, 150, or 200 or more such nucleic acids.
• the interferon-alpha homologues of the invention are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such interferon homologues in the context of the present invention to a patient are available, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
• Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
• Polypeptide compositions can be administered for any of the prophylactic, therapeutic, and diagnostic methods described herein by a number of routes including, but not limited to oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, vaginal, or rectal means, or by inhalation.
• Interferon homologue polypeptide compositions can also be administered via liposomes.
• Such administration routes and appropriate formulations are generally known to those of skill in the art.
• the interferon homologue polypeptide or nucleic acid, alone or in combination with other suitable components can also be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
• Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non- aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
• the formulations of packaged nucleic acid can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
• Parenteral administration and intravenous administration are prefened methods of administration.
• the routes of administration already in use for existing alpha interferon therapeutics or prophylactics, along with formulations in cunent use are prefened routes of administration and formulation for the alpha interferon homologue polypeptide and nucleic acids of the invention.
• Cells transduced with the interferon homologue nucleic acids as described above in the context of ex vivo or in vivo therapy can also be administered intravenously or parenterally as described above. It will be appreciated that the delivery of cells to subjects (e.g., human patients) is routine, e.g., delivery of cells to the blood via intravenous or intraperitoneal administration.
• the dose of interferon homologue polypeptide or nucleic acid of the invention administered to a subject (e.g., patient), in the context of the present invention is sufficient to effect a beneficial therapeutic or prophylactic response in the subject (e.g., patient) over time, or to inhibit infection by a pathogen, depending on the application.
• the dose will be determined by the efficacy of the particular vector, or formulation, and the activity interferon homologue employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
• the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, formulation, transduced cell type or the like in a particular patient.
• an effective amount of an interferon-alpha nucleic acid (e.g., DNA or mRNA) of the invention e.g., nucleic acid dosage
• an effective amount of an interferon-alpha nucleic acid (e.g., DNA or mRNA) of the invention will generally be in the range of, e.g., from about 0.05 microgram/kilogram (kg) to about 50 mg/kg, usually about 0.005-5 mg/kg.
• the effective amount of the nucleic acid e.g., nucleic acid dosage
• polpeptide e.g., polypeptide dosage
• the effective amount of the nucleic acid and/or polpeptide will vary in a manner apparent to those of ordinary skill in the art according to a number of factors, including the activity or potency of the polypeptide, the activity or potency of any nucleic acid construct (e.g., vector, promoter, expression system) to be administered, the disease or condition (e.g., particular cancer) to be treated, and the subject to which or whom the nucleic acid is delivered.
• nucleic acid construct e.g., vector, promoter, expression system
• nucleic acid dosage of, e.g., about 0.005mg/kg to about 5 mg/kg.
• Dosages for other polypeptides (and nucleic acids encoding them) having a known biological activity can be readily determined by those of skill in the art according to the factors noted above.
• Dosages used for other known interferon-alphas for particular diseases provide guidelines for determining dosage and treatment regimen for a nucleic acid or polypeptide of the invention.
• An effective amount of an interferon-alpha homologue polypeptide may be in the range of from about 1 microgram to about 1 milligram, and more typically from about 1 microgram to about 100 micrograms.
• a composition for use in therapeutic and prophylactic treatment methods of the invention described herein may comprise, e.g., a concentration of an interferon-alpha homologue nucleic acid (e.g., DNA or mRNA) of the invention of from about 0.1 microgram/milhliter (ml) to about 20 mg/ml and a pharmaceutically acceptable carrier (e.g., aqueous carrier).
• a concentration of an interferon-alpha homologue nucleic acid e.g., DNA or mRNA
• a pharmaceutically acceptable carrier e.g., aqueous carrier
• a composition for use in therapeutic and prophylactic treatment methods of the invention described herein may comprise, e.g., a concentration of an interferon-alpha homologue polypeptide of the invention in an amount as described above and herein and a pharmaceutically acceptable carrier (e.g., aqueous carrier).
• a pharmaceutically acceptable carrier e.g., aqueous carrier
• the physician evaluates circulating plasma levels, vector/cell/formulation/ interferon homologue toxicities, progression of the disease, and the production of anti- vector/interferon homologue antibodies.
• the dose administered, e.g., to a 70 kilogram patient will be in the range equivalent to dosages of cunently-used interferon-alpha therapeutic or prophylactic proteins, and doses of vectors or cells which produce interferon homologue sequences are calculated to yield an equivalent amount of interferon homologue nucleic acid or expressed protein.
• the vectors of this invention can supplement treatment of cancers and virally-mediated conditions by any known conventional therapy, including cytotoxic agents, nucleotide analogues (e.g., when used for treatment of HIV infection), biologic response modifiers, and the like.
• interferon homologues and transduced cells of the present invention can be administered at a rate determined by the LD-50 of the interferon homologue polypeptide or nucleic acid, vector, or transduced cell type, and the side- effects of the interferon homologue polypeptides or nucleic acids, vector or cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses.
• a subject e.g., patient
• blood samples are obtained prior to infusion, and saved for analysis.
• 1 X 10 6 and 1 X 10 12 transduced cells are infused intravenously over 60- 200 minutes.
• Vital signs and oxygen saturation by pulse oximetry are closely monitored.
• Blood samples are obtained 5 minutes and 1 hour following infusion and saved for subsequent analysis.
• Leukopheresis, transduction and reinfusion are optionally repeated every 2 to 3 months for a total of 4 to 6 treatments in a one year period. After the first treatment, infusions can be performed on a outpatient basis at the discretion of the clinician.
• the participant is monitored for at least 4, and preferably 8 hours following the therapy.
• Transduced cells are prepared for reinfusion according to established methods. See Abrahamsen et al. (1991) J. Clin. Apheresis 6:48-53; Carter et al. (1988) J. Clin. Arpheresis A: 113-117; Aebersold et al. (1988), J. Immunol. Methods 112:1-7; Muul et al. (1987) J. Immunol. Methods 101:171- 181 and Carter et al. (1987) Transfusion 27:362-365.
• the cells should number between 1 X 10 6 and 1 X 10 12 .
• the growth characteristics of cells vary from patient to patient and from cell type to cell type. About 72 hours prior to reinfusion of the transduced cells, an aliquot is taken for analysis of phenotype, and percentage of cells expressing the therapeutic or prophylactic agent.
• a subject e.g., patient
• a vector or transduced cell or protein formulation develops fevers, chills, or muscle aches
• he/she receives the appropriate dose of aspirin, ibuprofen, acetaminophen or other pain fever controlling drug.
• Subjects e.g., patients
• who experience reactions to the infusion such as fever, muscle aches, and chills are premedicated 30 minutes prior to the future infusions with either aspirin, acetaminophen, or, e.g., diphenhydramine.
• Meperidine is used for more severe chills and muscle aches that do not quickly respond to antipyretics and antihistamines. Cell infusion is slowed or discontinued depending upon the severity of the reaction.
• the present invention also includes methods of therapeutically or prophylactically treating a disease or disorder by administering in vivo or ex vivo one or more nucleic acids or polypeptides of the invention described above (or compositions comprising a pharmaceutically acceptable excipient and one or more such nucleic acids or polypeptides) to a subject, including, e.g., a mammal, including, e.g., a human, primate, mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, sheep; or a non-mammalian vertebrate such as a bird (e.g., a chicken or duck) or a fish, or invertebrate.
• a mammal including, e.g., a human, primate, mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, sheep; or a non-mammalian vertebrate such as a bird (e
• one or more cells or a population of cells of interest of the subject e.g., tumor cells, tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.
• a polypeptide of the invention that is effective in prophylactically or therapeutically treating the disease, disorder, or other condition.
• the contacted cells are then returned or delivered to the subject to the site from which they were obtained or to another site (e.g., including those defined above) of interest in the subject to be treated.
• the contacted cells may be grafted onto a tissue, organ, or system site (including all described above) of interest in the subject using standard and well-known grafting techniques or, e.g., delivered to the blood or lymph system using standard delivery or transfusion techniques.
• the invention also provides in vivo methods in which one or more cells or a population of cells of interest of the subject are contacted directly or indirectly with an amount of a polypeptide of the invention effective in prophylactically or therapeutically treating the disease, disorder, or other condition.
• the polypeptide is typically administered or transfened directly to the cells to be treated or to the tissue site of interest (e.g., tumor cells, tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) by any of a variety of formats, including topical administration, injection (e.g., by using a needle or syringe), or vaccine or gene gun delivery, pushing into a tissue, organ, or skin site.
• the tissue site of interest e.g., tumor cells, tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.
• the polypeptide can be delivered, for example, intramuscularly, intradermally, subdermally, subcutaneously, orally, intraperitoneally, intrathecally, intravenously, or placed within a cavity of the body (including, e.g., during surgery), or by inhalation or vaginal or rectal administration.
• the polypeptide is typically administered or transfened indirectly to the cells to be treated or to the tissue site of interest, including those described above (such as, e.g., skin cells, organ systems, lymphatic system, or blood cell system, etc.), by contacting or administering the polypeptide of the invention directly to one or more cells or population of cells from which treatment can be facilitated.
• tumor cells within the body of the subject can be treated by contacting cells of the blood or lymphatic system, skin, or an organ with a sufficient amount of the polypeptide such that delivery of the polypeptide to the site of interest (e.g., tissue, organ, or cells of interest or blood or lymphatic system within the body) occurs and effective prophylactic or therapeutic treatment results.
• site of interest e.g., tissue, organ, or cells of interest or blood or lymphatic system within the body
• Such contact, administration, or transfer is typically made by using one or more of the routes or modes of administration described above.
• the invention provides ex vivo methods in which one or more cells of interest or a population of cells of interest of the subject (e.g., tumor cells, tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) are obtained or removed from the subject and transformed by contacting said one or more cells or population of cells with a polynucleotide construct comprising a target nucleic acid sequence of the invention that encodes a biologically active polypeptide of interest (e.g., a polypeptide of the invention) that is effective in prophylactically or therapeutically treating the disease, disorder, or other condition.
• a polynucleotide construct comprising a target nucleic acid sequence of the invention that encodes a biologically active polypeptide of interest (e.g., a polypeptide of the invention) that is effective in prophylactically
• the one or more cells or population of cells is contacted with a sufficient amount of the polynucleotide construct and a promoter controlling expression of said nucleic acid sequence such that uptake of the polynucleotide construct (and promoter) into the cell(s) occurs and sufficient expression of the target nucleic acid sequence of the invention results to produce an amount of the biologically active polypeptide effective to prophylactically or therapeutically treat the disease, disorder, or condition.
• the polynucleotide construct may include a promoter sequence (e.g., CMV promoter sequence) that controls expression of the nucleic acid sequence of the invention and/or, if desired, one or more additional nucleotide sequences encoding at least one or more of another polypeptide of the invention, a cytokine, adjuvant, or co-stimulatory molecule, or other polypeptide of interest.
• a promoter sequence e.g., CMV promoter sequence
• additional nucleotide sequences encoding at least one or more of another polypeptide of the invention, a cytokine, adjuvant, or co-stimulatory molecule, or other polypeptide of interest.
• the transformed cells are returned, delivered, or transfened to the subject to the tissue site or system from which they were obtained or to another site (e.g., tumor cells, tumor tissue sample, organ cells, blood cells, cells of the skin, lung, heart, muscle, brain, mucosae, liver, intestine, spleen, stomach, lymphatic system, cervix, vagina, prostate, mouth, tongue, etc.) to be treated in the subject.
• the cells may be grafted onto a tissue, skin, organ, or body system of interest in the subject using standard and well-known grafting techniques or delivered to the blood or lymphatic system using standard delivery or transfusion techniques.
• Such delivery, administration, or transfer of transformed cells is typically made by using one or more of the routes or modes of administration described above.
• Expression of the target nucleic acid occurs naturally or can be induced (as described in greater detail below) and an amount of the encoded polypeptide is expressed sufficient and effective to treat the disease or condition at the site or tissue system.
• the invention provides in vivo methods in which one or more cells of interest or a population of cells of the subject (e.g., including those cells and cells systems and subjects described above) are transformed in the body of the subject by contacting the cell(s) or population of cells with (or administering or transferring to the cell(s) or population of cells using one or more of the routes or modes of administration described above) a polynucleotide construct comprising a nucleic acid sequence of the invention that encodes a biologically active polypeptide of interest (e.g., a polypeptide of the invention) that is effective in prophylactically or therapeutically treating the disease, disorder, or other condition.
• a polynucleotide construct comprising a nucleic acid sequence of the invention that encodes a biologically active polypeptide of interest (e.g., a polypeptide of the invention) that is effective in prophylactically or therapeutically treating the disease, disorder, or other condition.
• the polynucleotide construct can be directly administered or transfened to cell(s) suffering from the disease or disorder (e.g., by direct contact using one or more of the routes or modes of administration described above).
• the polynucleotide construct can be indirectly administered or transfened to cell(s) suffering from the disease or disorder by first directly contacting non-diseased cell(s) or other diseased cells using one or more of the routes or modes of administration described above with a sufficient amount of the polynucleotide construct comprising the nucleic acid sequence encoding the biologically active polypeptide, and a promoter controlling expression of the nucleic acid sequence, such that uptake of the polynucleotide construct (and promoter) into the cell(s) occurs and sufficient expression of the nucleic acid sequence of the invention results to produce an amount of the biologically active polypeptide effective to prophylactically or therapeutically treat the disease or disorder, and whereby the polynucleotide construct or the resulting expressed polypeptide is transfened
• the polynucleotide construct may include a promoter sequence (e.g., CMV promoter sequence) that controls expression of the nucleic acid sequence and/or, if desired, one or more additional nucleotide sequences encoding at least one or more of another polypeptide of the invention, a cytokine, adjuvant, or co-stimulatory molecule, or other polypeptide of interest.
• a promoter sequence e.g., CMV promoter sequence
• additional nucleotide sequences encoding at least one or more of another polypeptide of the invention, a cytokine, adjuvant, or co-stimulatory molecule, or other polypeptide of interest.
• compositions comprising an excipient and the polypeptide or nucleic acid of the invention can be administered or delivered.
• a composition comprising a pharmaceutically acceptable excipient and a polypeptide or nucleic acid of the invention is administered or delivered to the subject as described above in an amount effective to treat the disease or disorder.
• the amount of polynucleotide administered to the cell(s) or subject can be an amount sufficient that uptake of said polynucleotide into one or more cells of the subject occurs and sufficient expression of said nucleic acid sequence results to produce an amount of a biologically active polypeptide effective to enhance an immune response in the subject, including an immune response induced by an immunogen (e.g., antigen).
• an immunogen e.g., antigen
• the amount of polypeptide administered to cell(s) or subject can be an amount sufficient to enhance an immune response in the subject, including that induced by an immunogen (e.g., antigen).
• the expression of the polynucleotide construct can be induced by using an inducible on- and off-gene expression system.
• on- and off-gene expression systems include the Tet-OnTM Gene Expression System and Tet-OflTM Gene Expression System (see, e.g., Clontech Catalog 2000, pg. 110-111 for a detailed description of each such system), respectively.
• Tet-OnTM Gene Expression System and Tet-OflTM Gene Expression System (see, e.g., Clontech Catalog 2000, pg. 110-111 for a detailed description of each such system), respectively.
• Other controllable or inducible on- and off-gene expression systems are known to those of ordinary skill in the art.
• expression of the target nucleic of the polynucleotide construct can be regulated in a precise, reversible, and quantitative manner.
• Gene expression of the target nucleic acid can be induced, for example, after the stable transfected cells containing the polynucleotide construct comprising the target nucleic acid are delivered or transfened to or made to contact the tissue site, organ or system of interest.
• Such systems are of particular benefit in treatment methods and formats in which it is advantageous to delay or precisely control expression of the target nucleic acid (e.g., to allow time for completion of surgery and/or healing following surgery; to allow time for the polynucleotide construct comprising the target nucleic acid to reach the site, cells, system, or tissue to be treated; to allow time for the graft containing cells transformed with the construct to become inco ⁇ orated into the tissue or organ onto or into which it has been spliced or attached, etc.)
• INTEGRATED SYSTEMS INTEGRATED SYSTEMS
• the present invention provides computers, computer readable media and integrated systems comprising character strings conesponding to the sequence information herein for the polypeptides and nucleic acids herein, including, e.g., those sequences listed herein and the various silent substitutions and conservative substitutions thereof.
• GOs genetic algorithms
• homology determination methods have been designed for comparative analysis of sequences of biopolymers, for spell-checking in word processing, and for data retrieval from various databases.
• models that simulate annealing of complementary homologous polynucleotide strings can also be used as a foundation of sequence alignment or other operations typically performed on the character strings conesponding to the sequences herein (e.g., word-processing manipulations, construction of figures comprising sequence or subsequence character strings, output tables, etc.).
• An example of a software package with GOs for calculating sequence similarity or homology is BLAST, which can be adapted to the present invention by inputting character strings conesponding to the sequences herein.
• standard desktop applications such as word processing software (e.g., Microsoft WordTM or Corel WordPerfectTM) and database software (e.g., spreadsheet software such as Microsoft ExcelTM, Corel Quattro ProTM, or database programs such as Microsoft AccessTM or ParadoxTM) can be adapted to the present invention by inputting a character string conesponding to the interferon alpha homologues of the invention (either nucleic acids or proteins, or both).
• the integrated systems can include the foregoing software having the appropriate character string information, e.g., used in conjunction with a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh or LINUX system) to manipulate strings of characters.
• specialized alignment programs such as BLAST can also be inco ⁇ orated into the systems of the invention for alignment of nucleic acids or proteins (or conesponding character strings).
• Integrated systems for analysis in the present invention typically include a digital computer with GO software for aligning sequences, as well as data sets entered into the software system comprising any of the sequences herein.
• the computer can be, e.g., a PC (Intel x86 or Pentium chip- compatible DOSTM, OS2TM WINDOWSTM WINDOWS NTTM, WINDOWS95TM, WINDOWS98TM LINUX based machine, a MACINTOSHTM, Power PC, or a UNIX based (e.g., SUNTM work station) machine) or other commercially common computer which is known to one of skill.
• Software for aligning or otherwise manipulating sequences is available, or can easily be constructed by one of skill using a standard programming language such as Visualbasic, Fortran, Basic, Java, or the like.
• Any controller or computer optionally includes a monitor which is often a cathode ray tube ("CRT") display, a flat panel display (e.g., active matrix liquid crystal display, liquid crystal display), or others.
• Computer circuitry is often placed in a box which includes numerous integrated circuit chips, such as a microprocessor, memory, interface circuits, and others.
• the box also optionally includes a hard disk drive, a floppy disk drive, a high capacity removable drive such as a writeable CD-ROM, and other common peripheral elements.
• Inputting devices such as a keyboard or mouse optionally provide for input from a user and for user selection of sequences to be compared or otherwise manipulated in the relevant computer system.
• the computer typically includes appropriate software for receiving user instructions, either in the form of user input into a set parameter fields, e.g., in a GUI, or in the form of preprogrammed instructions, e.g., preprogrammed for a variety of different specific operations.
• the software then converts these instructions to appropriate language for instructing the operation of the fluid direction and transport controller to cany out the desired operation.
• the software can also include output elements for controlling nucleic acid synthesis (e.g., based upon a sequence or an alignment of a sequences herein) or other operations which occur downstream from an alignment or other operation performed using a character string conesponding to a sequence herein.
• the invention provides an integrated system comprising a computer or computer readable medium comprising a database having one or more sequence records. Each of the sequence records comprises one or more character strings conesponding to a nucleic acid or polypeptide or protein sequence selected from SEQ ED NO: 1 to SEQ ED NO:85.
• the integrated system further comprises a use input interface allowing a use to selectively view the one or more sequence records.
• the computer or computer readable medium comprises an alignment instruction set that aligns the character strings with one or more additional character strings conesponding to a nucleic acid or polypeptide or protein sequence.
• One such integrated system includes an instruction set that comprises at least one of the following: a local homology comparison determination, a homology alignment determination, a search for similarity determination, and a BLAST determination.
• the system further comprises a readable output element that displays an alignment produced by the alignment instruction set.
• the computer or computer readable medium further comprises an instruction set that translates at least one nucleic acid sequence which comprises a sequence selected from SEQ ED NO:l to SEQ ID NO:35 or SEQ ID NO:72 to SEQ ID NO:78 into an amino acid sequence.
• the instruction set may select the nucleic acid by applying a codon usage instruction set or an instruction set which determines sequence identity to a test nucleic acid sequence.
• Each of the sequence records comprises at least one character string conesponding to SEQ ID NO:l to SEQ ID NO:85.
• the method comprises determining at least one character string conesponding to one or more of SEQ ID NO: 1 to SEQ ED NO: 85 or a subsequence thereof; determining which of the at least one character string of the list are selected by a user; and displaying each of the selected character strings, or aligning each of the selected character strings with an additional character string.
• the method may further comprise displaying an alignment of each of the selected character strings with an additional character string and/or displaying the list.
• kits embodying the methods, composition, systems and apparatus herein optionally comprise one or more of the following: (1) an apparatus, system, system component or apparatus component as described herein; (2) instructions for practicing the methods described herein, and/or for operating the apparatus or apparatus components herein and/or for using the compositions herein; (3) one or more alpha interferon homologue compositions (such as e.g., compositions comprising at least one interferon alpha homologue nucleic acid or polypeptide or fragment thereof, cell, vector, etc., of the invention) or components (interferon alpha homologue nucleic acid or polypeptide or fragment thereof, cell, vector, etc., of the invention); (4) a container for holding one or more aspects of the invention, including such components or compositions, and (5) packaging materials.
• alpha interferon homologue compositions such as e.g., compositions comprising at least one interferon alpha homologue nucleic acid or polypeptide or fragment thereof, cell, vector, etc., of the invention
• components interferon al
• the present invention provides for the use of any apparatus, apparatus component, composition or kit herein, for the practice of any method or assay herein, and/or for the use of any apparatus or kit to practice any assay or method herein.
• Fragments (25-60 base pairs (bp) in length) of about 20 human interferon- alpha subspecies genes were prepared by PCR amplification and DNAse treatment, and recombined essentially as described in Crameri A. et al. (1998; Nature 15:288-291), to produce shuffled interferon-alpha mature coding sequences.
• Expression libraries were prepared by subcloning shuffled interferon-alpha mature coding sequences into an E. coli secretion vector.
• Shuffled interferon polypeptides were expressed as mature proteins fused at the C-termini to an E tag (Amersham-Pharmacia) to facilitate quantitation and purification from the periplasmic space.
• E. coli transformants were picked using a robotic colony picker (Q-Bot, Genetix Pharmaceuticals) into microtiter plates, and periplasmic extracts were prepared.
• Periplasmic extracts were assayed for antiproliferative activity on a human Daudi cell line as described by Scarozza, A.M. et al. (1992) J. Interferon Res. 12:35-42. Clones exhibiting antiproliferative activity in the Daudi assay were re- screened and expression levels determined by Western blot using an anti-E tag antibody (Amersham-Pharmacia). Clones exhibiting highest activity normalized to expression levels were selected for sequencing and were also utilized as substrates for additional rounds of shuffling and screening as described above.
• Clones from the first and second rounds of shuffling having relatively high antiproliferative activity by the Daudi assay were subcloned into a CHO expression vector (pDEI-1011) in which the E-tag/6-His tag (Amersham-Pharmacia) is fused to the C- terminus of the shuffled interferons.
• Clones were transfected into CHO cells and stable cell lines were selected with 1 mg/ml G418.
• CHO-expressed mature interferons were purified on anti-E tag Sepharose column (Amersham-Pharmacia) and quantitated by a Bradford assay (Biorad).
• WISH cells were seeded to a density of 6 x 10 4 cells/well in 96-well plates in 100 ul RPMI medium (Gibco-BRL) supplemented with 10% fetal calf serum, penicillin
• Fig. 2 shows the antiproliferative activity and the antiviral activity of exemplary interferon homologues of the invention, in comparison with interferon alpha-2a and interferon-alpha Conl .
• the graph shows the number of Units activity per milligram of homologue (Y axis) for a set of exemplary interferon alpha homologues, each of which is designated with a "name" on the X axis.
• EXAMPLE 2 7N VITRO CANCER CELL LINE SCREEN
• the 60 human cancer cell lines used include leukemias, melanomas, and cancers of the lung, colon, brain, ovary, breast, prostate, central nervous system, renal system, and kidney. Human tumor cell lines were grown according to procedures outlined in Monks") and http://dtp.nci. gov./branches/btb/i vclsp.html.
• cells were inoculated into 96 well microtiter plates at densities ranging from about 5,000 to about 40,000 cells/well, depending on the growth properties of the particular cell line.
• the microtiter plates were incubated for 24 hours (h) at 37 degrees C prior to addition of test samples (e.g., interferon homologues of the invention or control interferons).
• test samples e.g., interferon homologues of the invention or control interferons.
• TCA trichloroacetic acid
• interferon samples (affinity-purified from CHO cell supernatants) were added in five 10-fold serial dilutions ranging from 10 " ° '8 to 10 "4 8 ⁇ g/ml. Following sample addition, the plates were incubated for an additional 6 days. The assay was terminated by addition of TCA.
• Cell population was determined by measuring cellular protein in a quantitative protein dye-binding assay. Sulforhodamine B solution (100 ⁇ l) at 0.4 % (w/v) in 1% acetic acid was added to each well, followed by incubation for 10 minutes at room temperature. Unbound dye was removed by washing five times with 1% acetic acid and the plates air- dried. Protein-bound dye was solubilized with 10 milliMolar (mM) Tris, and the absorbance read at 515 nanometer (nm) on an automated plate reader .
• mM milliMolar
• GI50 growth inhibition of 50%
• concentration of interferon test sample at which cell growth is inhibited by 50% is measured by a 50% reduction in the net protein/polypeptide increase in the interferon test sample as compared to that observed in the control cells (no test sample) at the end of the incubation period.
• TGI total growth inhibition
• LC50 is the concentration of interferon test sample at which a 50% reduction in the measured amount of cellular protein at the end of the incubation as compared to that at the beginning of the incubation period is observed, indicating a net loss of cells following interferon test sample addition.
• CH2.2 and CH2.3 were used in this study.
• CH2.2 and CH2.3 were shown to have about 138,000-fold and about 206,00-fold higher activity, respectively, than human interferon-alpha 2a, and about 2.5-fold and about 1.6-fold higher activity than native mouse interferon-alpha 4, in the in vitro mouse cell antiviral assay (Chang et al, supra).
• mice received subcutaneous doses of either phosphate buffered saline (PBS), interferon-alpha homologue CH2.2, interferon-alpha homologue CH2.3, murine IFN-alpha 4, or human interferon-alpha 2a, in daily subcutaneous doses of 2, 10, or 50 ⁇ g (total volume of 50 ⁇ l) for four consecutive days.
• PBS phosphate buffered saline
• interferon-alpha homologue CH2.2, interferon-alpha homologue CH2.3, murine IFN-alpha 4, or human interferon-alpha 2a in daily subcutaneous doses of 2, 10, or 50 ⁇ g (total volume of 50 ⁇ l) for four consecutive days.
• the mice were exposed to a lethal intranasal dose (ten times the LC50) of vesicular stomatitis virus (VSV). Data is expressed as the number of mice which survive to day 21.
• VSV vesicular stomatitis virus
• Fig. 5 shows that both of the mouse-optimized interferon-alpha homologues, CH2.2 and CH2.3, were as effective or more effective than native murine interferon Mu-IFN alpha 4 in protecting mice from VSV.
• human IFN-alpha 2a was nearly completely ineffective in protecting mice from the virus.
• the in vivo efficacy of the interferon-alpha homologues of the invention conelates remarkably well with the antiviral activities observed in the in vitro assays.

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