Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules

• US 20070172459A1
• Filed: 02/13/2007
• Published: 07/26/2007
• Est. Priority Date: 09/09/2002
• Status: Active Grant

• Current Assignee: Lila Drittanti, Manuel Vega, Rene Gantier, Thierry Guyon
• Original Assignee: HanAll Biopharma Co. Ltd.
• Inventors: Guyon, Thierry, Drittanti, Lila, Vega, Manuel, Gantier, Rene
• Granted Patent: US 7,998,469 B2
• Time in Patent Office: Days
• US Class Current: 424/85.500
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/53 Colony-stimulating factor [CSF]
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/54 Interleukins [IL]
• CPC Class Codes: C07K 14/555 : Interferons [IFN]
• CPC Class Codes: C07K 14/575 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/61 : Growth hormone [GH], i.e. somatotropin

Abstract

Compositions of modified cytokines and uses thereof generated using processes and systems for the high throughput directed evolution of peptides and proteins, particularly cytokines that act in complex biological settings, are provided. Also provided are modified cytokines formulated for oral delivery and uses thereof to treat diseases and conditions mediated by cytokines.

Claims

1. A modified interferon beta (IFN beta) cytokine, comprising one or more amino acid replacements in its sequence of amino acid residues, wherein:

the modified IFN beta cytokine exhibits increased resistance to proteolysis compared to the unmodified IFN beta cytokine that does not comprise the one or more amino acid replacements;

the one or more amino acid replacements and positions thereof are selected from among replacement of;

M1V, M1I, M1T, M1A, M1D, M1E, M1K, M1N, M1R, M1S, M1Q, L5V, L5I, L6K, L6N, L6Q, L6R, L6C, F8I, F8V, L9V, L9I, L9T, L9Q, L9H, L9A, L9S, Q10S, Q10T, R11H, R11Q, S13Q, N14D, N14E, N14K, N14Q, N14R, N14S, N14T, F15I, F15V, Q16N, Q16R, Q16S, Q16T, C17K, C17Q, C17R, K19T, K19S, K19H, L20N, L20Q, L20R, L20S, L20T, Q23R, N25H, N25S, N25Q, R27H, R27Q, L28V, L28I, E29Q, Y30I, L32V, L32I, K33T, K33S, K33H, R35H, R35Q, M36V, M36I, D39N, D39H, D39G, E42N, E42H, K45N, K45T, K45S, K45H, L47V, L47I, K52N, K52T, K52S, K52H, F67I, F67V, R71H, R71Q, D73H, D73G, E81H, I83Q, I83S, I83T, E85H, N86D, N86R, N86Q, N86S, N86T, L87Q, L87S, L87T, N90Q, N90S, N90T, V91Q, V91S, V91T, Y92I, Q94S, Q94T, I95K, I95N, I95Q, I95R, I95S, I95T, H97N, H97Q, H97R, H97S, H97T, L98D, L98E, L98K, L98N, L98Q, L98R, L98C, K99T, K99S, K99H, V101D, V101E, V101K, V101N, V101Q, V101R, V101S, V101T, V101C, E103H, K105T, K105S, K105H, E107N, E107H, K108N, K108T, K108S, K108H, E109N, E109H, D110H, D110G, F111I, F111V, R113H, L116V, L116I, L120V, L120I, K123N, K123T, K123S, K123H, R124H, R124Q, R128H, R128Q, L130V, L130I, K134N, K134T, K134S, K134H, K136N, K136T, K136S, K136H, E137H, Y138H, Y138I, R152H, R152Q, Y155H, Y155I, R159H, R159Q, Y163H, Y163I, R165H and R165Q; and

the one or more amino acid replacements occur in a mature IFN beta cytokine having the sequence set forth in SEQ ID NO;

196 or in a sequence-related IFN beta cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

196.

2. The modified IFN beta cytokine of claim 1, wherein:

the one or more amind acid replacements and positions thereof are selected from among replacement of;

M1V, M1I, M1T, M1A, M1D, M1E, M1K, M1N, M1R, M1S, M1Q, L5V, L5I, F8I, F8V, Q10S, Q10T, R11H, R11Q, S13Q, N14E, N14K, N14Q, N14R, N14S, N14T, F15I, F15V, Q16N, Q16R, Q16S, Q16T, K19T, K19S, K19H, Q23R, N25H, N25S, N25Q, R27H, R27Q, Y30I, K33S, R35Q, D39N, D39H, E42N, E42H, K45N, K45T, K45S, K45H, L47V, L47I, K52S, F67I, F67V, R71H, R71Q, D73H, E81H, E85H, N86D, N86R, N86Q, N86S, N86T, N90Q, N90S, Y92I, Q94S, Q94T, H97N, H97Q, H97R, H97S, H97T, K99T, K99S, K99H, E103H, E107N, E109N, E109H, D110 G, F111I, F111V, R113H, L120V, L1201, K123N, K123S, K123H, R124H, R124Q, R128Q, L130V, L130I, K134N, K134T, K134S, K136N, K136S, K136H, E137H, Y138I, R152H, R152Q, Y155I, Y163H, Y163I, R165H and R165Q; and

the one or more amino acid replacements occur in a mature IFN beta cytokine having the sequence set forth in SEQ ID NO;

196 or in a sequence-related IFN beta cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

196.

3. The modified IFN beta cytokine of claim 1, wherein:

the one or more amino acid replacements and positions thereof are selected from among replacement of;

M1V, M1I, M1T, M1A, M1D, M1E, M1K, M1N, M1R, M1S, M1Q, L5V, L5I, L6K, L6N, L6Q, L6R, L6C, F8I, F8V, L9V, L9I, L9T, L9Q, L9H, L9A, L9S, Q10S, Q10T, R11H, R11Q, S13Q, N14D, N14E, N14K, N14Q, N14R, N14S, N14T, F15I, F15V, Q16N, Q16R, Q16S, Q16T, C17K, C17Q, C17R, K19T, K19S, K19H, L20N, L20Q, L20R, L20S, L20T, Q23R, N25H, N25S, N25Q, R27H, R27Q, L28V, L28I, E29Q, Y30I, L32V, L32I, K33T, K33S, K33H, R35H, R35Q, M36V, M36I, D39H, D39G, K45T, K45S, K45H, K52T, K52S, K52H, D73H, D73G, I83Q, I83S, I83T, E85H, N86D, N86R, N86Q, N86S, N86T, L87Q, L87S, L87T, N90Q, N90S, N90T, V91Q, V91 S, V91T, Y92I, Q94S, Q94T, I95K, I95N, I95Q, I95R, I95S, I95T, H97N, H97Q, H97R, H97S, H97T, L98D, L98E, L98K, L98N, L98Q, L98R, L98C, K99T, K99S, K99H, V101D, V101E, V101K, V101N, V101Q, V101R, V101S, V101T, V101C, E103H, K105T, K105S, K105H, K108T, K108S, K108H, D101H, D101G, K123T, K123S, K123H, K134T, K134S, K134H, K136T, K136S, K136H, Y138H, Y138I, R152H, R152Q, Y155H, Y155I, R159H and R159Q; and

the one or more amino acid replacements occur in a mature IFN beta cytokine having the sequence set forth in SEQ ID NO;

196 or in a sequence-related IFN beta cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

196.

4. The modified IFN beta cytokine of claim 1, wherein:

the one or more amino acid replacements and positions thereof are selected from among replacement of;

D39N, E42N, E42H, K45N, L47V, L47I, K52N, F67I, F67V, R71H, R71Q, E81H, E107N, E107H, K108N, E109N, E109H, F111, F111V, R113H, L116V, L116I, L120V, L1201, K123N, R124H, R124Q, R128H, R128Q, L130V, L130I, K134N, K136N, E137H, Y163H, Y163I, R165H and R165Q; and

the one or more amino acid replacements occur in a mature IFN beta cytokine having the sequence set forth in SEQ ID NO;

196 or in a sequence-related IFN beta cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

196.

5. The modified IFN beta cytokine of claim 1, wherein the unmodified IFN beta cytokine contains the amino acids residues having the sequence set forth in SEQ ID NO:

196 or 197.

6. The IFN beta cytokine of claim 1 that comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the amino acid replacements in its sequence of amino acid residues.

7. The IFN beta cytokine of claim 1 that comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid replacements in its sequence of amino acid residues.

8. The IFN beta cytokine of claim 1, further comprising one or more additional amino acid replacements in its sequence of amino acids, wherein:

the one or more amino acid replacements and positions thereof are selected from among replacement of;

M1V, M1I, M1T, M1A, M1D, M1E, M1K, M1N, M1R, M1S, M1Q, M1C, L5V, L5I, L5T, L5Q, L5H, L5A, L5D, L5E, L5K, L5R, L5N, L5S, L6D, L6E, L6K, L6N, L6Q, L6R, L6S, L6T, L6C, F8I, F8V, F8D, F8E, F8K, F8R, L9V, L9I, L9T, L9Q, L9H, L9A, L9D, L9E, L9K, L9N, L9R, L9S, Q10D, Q10E, Q10K, Q10N, Q10R, Q10S, Q10T, Q10C, R11H, R11Q, S12D, S12E, S12K, S12R, S13D, S13E, S13K, S13N, S13Q, S13R, S13T, S13C, N14D, N14E, N14K, N14Q, N14R, N14S, N14T, F15I, F15V, F15D, F15E, F15K, F15R, Q16D, Q16E, Q16K, Q16N, Q16R, Q16S, Q16T, Q16C, C17D, C17E, C17K, C17N, C17Q, C17R, C17S, C17T, C17L, K19Q, K19T, K19S, K19H, L20N, L20Q, L20R, L20S, L20T, L20D, L20E, L20K, W22S, W22H, W22D, W22E, W22K, W22R, Q23D, Q23E, Q23K, Q23R, L24D, L24E, L24K, L24R, N25H, N25S, N25Q, R27H, R27Q, L28V, L28I, L28T, L28Q, L28H, L28A, E29Q, E29H, Y30H, Y30I, L32V, L32I, L32T, L32Q, L32H, L32A, K33Q, K33T, K33S, K33H, R35H, R35Q, M36V, M36I, M36T, M36Q, M36A, D39Q, D39N, D39H, D39G, E42Q, E42N, E42H, K45Q, K45N, K45T, K45S, K45H, L47V, L47I, L47T, L47Q, L47H, L47A, K52Q, K52N, K52T, K52S, K52H, F67I, F67V, R71H, R71Q, D73Q, D73N, D73H, D73G, G78D, G78E, G78K, G78R, W79D, W79E, W79K, W79R, N80D, N80E, N80K, N80R, E81Q, E81N, E81H, T82D, T82E, T82K, T82R, I83D, I83E, I83K, I83R, I83N, I83Q, I83S, I83T, E85Q, E85H, N86D, N86E, N86K, N86R, N86Q, N86S, N86T, L87D, L87E, L87K, L87R, L87N, L87Q, L87S, L87T, A89D, A89E, A89K, A89R, N90D, N90E, N90K, N90Q, N90R, N90S, N90T, N90C, V91D, V91E, V91K, V91N, V91Q, V91R, V91S, V91T, V91C, Y92H, Y92I, Q94D, Q94E, Q94K, Q94N, Q94R, Q94S, Q94T, Q94C, I95D, I95E, I95K, I95N, I95Q, I95R, I95S, I95T, H97D, H97E, H97K, H97N, H97Q, H97R, H97S, H97T, H97C, L98D, L98E, L98K, L98N, L98Q, L98R, L98S, L98T, L98C, K99Q, K99T, K99S, K99H, V101D, V101E, V101K, V101N, V101Q, V101R, V101S, V101T, V101C, E103Q, E103H, E104Q, E104H, K105Q, K105T, K105S, K105H, E107Q, E107N, E107H, K108Q, K108N, K108T, K108S, K108H, E109Q, E109N, E109H, D110Q, D110N, D110H, D110G, F111I, F111V, R113H, R113Q, L116V, L116I, L116T, L116Q, L116H, L116A, L120V, L120I, L120T, L120Q, L120H, L120A, K123Q, K123N, K123T, K123S, K123H, R124H, R124Q, R128H, R128Q, L130V, L130I, L130T, L130Q, L130H, L130A, K134Q, K134N, K134T, K134S, K134H, K136Q, K136N, K136T, K136S, K136H, E137Q, E137N, E137H, Y138H, Y138I, R152H, R152Q, Y155H, Y155I, R159H, R159Q, Y163H, Y163I, R165H and R165Q; and

the one or more amino;

acid replacements occur in a mature IFN beta cytokine having the sequence set forth in SEQ ID NO;

196 or in a sequence-related IFN beta cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

196.

9. The IFN beta cytokine of claim 1, wherein only the primary amino acid sequence is modified, and the IFN beta cytokine exhibits increased resistance to proteolysis.

10. The IFN beta cytokine of claim 1 that exhibits increased stability compared to the unmodified IFN beta cytokine.

11. The IFN beta cytokine of claim 10, wherein the IFN beta cytokine exhibits increased stability to proteases, human blood lysate or human serum.

12. The IFN beta cytokine of claim 1 that exhibits increased protein half-life in vitro or in vivo compared to the unmodified IFN beta cytokine.

13. The IFN beta cytokine of claim 1 that exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract.

14. The IFN beta cytokine of claim 1 that exhibits increased resistance to proteolysis by a protease in the serum.

15. The IFN beta cytokine of claim 1, wherein increased resistance to proteolysis is due to replacement of one or more amino acids at target positions in an unmodified IFN beta cytokine that increase resistance of the IFN beta cytokine to digestion by a protease.

16. The IFN beta cytokine of claim 1 that exhibits increased antiviral and/or anti-proliferative activity compared to the unmodified IFN beta cytokine.

17. The IFN beta cytokine of claim 1 that exhibits comparable antiviral and/or anti-proliferative activity compared to the unmodified IFN beta cytokine.

18. The IFN beta cytokine of claim 1 that exhibits decreased antiviral and/or anti-proliferative activity compared to the unmodified IFN beta cytokine.

20. A nucleic acid molecule encoding a modified IFN beta cytokine of claim 1.

29. A pharmaceutical composition, comprising an IFN beta cytokine of claim 1.

30. The pharmaceutical composition of claim 29, further comprising a pharmaceutically acceptable,carrier or excipient.

31. The pharmaceutical composition of claim 30, wherein the pharmaceutically acceptable carrier or excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant, and a wetting agent.

32. The pharmaceutical composition of claim 29, further comprising a pharmaceutically acceptable additive.

33. The pharmaceutical composition of claim 32, wherein the pharmaceutically acceptable additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle, and a preservative.

34. The pharmaceutical composition of claim 29, wherein the composition is in the form of a liquid, a solution, a suspension, an aerosol, a tablet, a lozenge or a capsule.

35. The pharmaceutical composition of claim 34, wherein the composition is in the form of a tablet or a capsule.

36. The pharmaceutical composition of claim 29, formulated for oral, parenteral, intravenous, intradermal, subcutaneous, buccal, inhalation, intramuscular, rectal or topical administration.

37. The pharmaceutical composition of claim 36, formulated for oral administration.

38. The pharmaceutical composition of claim 37, wherein the pharmaceutical composition is formulated for oral administration to the mouth or gastrointestinal tract.

39. The pharmaceutical composition of claim 29, wherein the pharmaceutical composition is formulated for controlled-release of the IFN beta cytokine.

63. A method, comprising treating a subject by administering the pharmaceutical composition of claim 29, wherein the subject has a disease or condition that is treated by administration of an IFN beta cytokine.

64. The method of claim 63, wherein in the disease or condition is selected from among infectious diseases, allergies, microbial diseases, pregnancy related diseases, bacterial diseases, heart diseases, viral diseases, histological diseases, genetic diseases, blood related diseases, fungal diseases, adrenal diseases, cancers, liver diseases, autoimmune diseases, growth disorders, diabetes, neurodegenerative diseases, multiple sclerosis, Parkinson'"'"'s disease and Alzheimer'"'"'s disease.

65. The method of claim 64, wherein in the disease or condition is multiple sclerosis.

19. A modified interferon beta (IFN beta) cytokine, comprising two or more amino acid replacements in its sequence of amino acid residues, wherein:

the modified IFN beta cytokine exhibits increased resistance to proteolysis compared to the unmodified IFN beta cytokine that does not comprise the one or more amino acid replacements;

the two or more amino acid replacements and positions thereof are selected from among replacement of;

M1V, M1I, M1T, M1A, M1D, M1E, M1K, M1N, M1R, M1S, M1Q, M1C, L5V, L5I, L5T, L5Q, L5H, L5A, L5D, L5E, L5K, L5R, L5N, L5S, L6D, L6E, L6K, L6N, L6Q, L6R, L6S, L6T, L6C, F8I, F8V, F8D, F8E, F8K, F8R, L9V, L9I, L9T, L9Q, L9H, L9A, L9D, L9E, L9K, L9N, L9R, L9S, Q10D, Q10E, Q10K, Q10N, Q10R, Q10S, Q10T, Q10C, R11H, R11Q, S12D, S12E, S12K, S12R, S13D, S13E, S13K, S13N, S13Q, S13R, S13T, S13C, N14D, N14E, N14K, N14Q, N14R, N14S, N14T, F15I, F15V, F15D, F15E, F15K, F15R, Q16D, Q16E, Q16K, Q16N, Q16R, Q16S, Q16T, Q16C, C17D, C17E, C17K, C17N, C17Q, C17R, C17S, C17T, C17L, K19Q, K19T, K19S, K19H, L20N, L20Q, L20R, L20S, L20T, L20D, L20E, L20K, W22S, W22H, W22D, W22E, W22K, W22R, Q23D, Q23E, Q23K, Q23R, L24D, L24E, L24K, L24R, N25H, N25S, N25Q, R27H, R27Q, L28V, L28I, L28T, L28Q, L28H, L28A, E29Q, E29H, Y30H, Y30I, L32V, L32I, L32T, L32Q, L32H, L32A, K33Q, K33T, K33S, K33H, R35H, R35Q, M36V, M36I, M36T, M36Q, M36A, D39Q, D39N, D39H, D39G, E42Q, E42N, E42H, K45Q, K45N, K45T, K45S, K45H, L47V, L47I, L47T, L47Q, L47H, L47A, K52Q, K52N, K52T, K52S, K52H, F67I, F67V, R71H, R71Q, D73Q, D73N, D73H, D73G, G78D, G78E, G78K, G78R, W79D, W79E, W79K, W79R, N80D, N80E, N80K, N80R, E81Q, E81N, E81H, T82D, T82E, T82K, T82R, I83D, I83E, I83K, I83R, I83N, I83Q, I83S, I83T, E85Q, E85H, N86D, N86E, N86K, N86R, N86Q, N86S, N86T, L87D, L87E, L87K, L87R, L87N, L87Q, L87S, L87T, A89D, A89E, A89K, A89R, N90D, N90E, N90K, N90Q, N90R, N90S, N90T, N90C, V91D, V91E, V91K, V91N, V91Q, V91R, V91S, V91T, V91C, Y92H, Y92I, Q94D, Q94E, Q94K, Q94N, Q94R, Q94S, Q94T, Q94C, I95D, I95E, I95K, I95N, I95Q, I95R, I95S, I95T, H97D, H97E, H97K, H97N, H97Q, H97R, H97S, H97T, H97C, L98D, L98E, L98K, L98N, L98Q, L98R, L98S, L98T, L98C, K99Q, K99T, K99S, K99H, V101D, V101E, V101K, V101N, V101Q, V101R, V101S, V101T, V101C, E103Q, E103H, E104Q, E104H, K105Q, K105T, K105S, K105H, E107Q, E107N, E107H, K108Q, K108N, K108T, K108S, K108H, E109Q, E109N, E109H, D110Q, D110N, D110H, D110G, F111I, F111V, R113H, R113Q, L116V, L116I, L116T, L116Q, L116H, L116A, L120V, L120I, L120T, L120Q, L120H, L120A, K123Q, K123N, K123T, K123S, K123H, R124H, R124Q, R128H, R128Q, L130V, L130I, L130T, L130Q, L130H, L130A, K134Q, K34N, K134T, K134S, K134H, K136Q, K136N, K136T, K136S, K136H, E137Q, E137N, E137H, Y138H, Y138I, R152H, R152Q, Y155H, Y155I, R159H, R159Q, Y163H, Y163I, R165H and R165Q; and

the one or more amino acid replacements occur in a mature IFN beta cytokine having the sequence set forth in SEQ ID NO;

196 or in a sequence-related IFN beta cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

196.

21. A vector, comprising a nucleic acid molecule of claim 19.

22. A eukaryotic cell, comprising the nucleic acid molecule of claim 19.

23. A eukaryotic cell, comprising the vector of claim 21.

24. A collection of nucleic acid molecules, comprising a plurality of the molecules of claim 19.

25. A collection of nucleic acid molecules, comprising a plurality of the vectors of claim 21.

26. A method for expression of a modified IFN beta cytokine, comprising:

introducing a nucleic acid of claim 19 into a host; and

culturing the cell under conditions, whereby the encoded modified IFN beta cytokine is expressed.

27. The method of claim 26, further comprising isolating the modified IFN beta cytokine.

28. The method of claim 26, wherein the host cell is a eukaryotic host cell or a bacterial cell.

62. A pharmaceutical composition, comprising a nucleic acid molecule of claim 19.

69. A method, comprising treating a subject by administering the pharmaceutical composition of claim 62, wherein the subject has a disease or condition that is treated by administration of an IFN beta cytokine.

70. The method of claim 69, wherein in the disease or condition is selected from among infectious diseases, allergies, microbial diseases, pregnancy related diseases, bacterial diseases, heart diseases, viral diseases, histological diseases, genetic diseases, blood related diseases, fungal diseases, adrenal diseases, cancers, liver diseases, autoimmune diseases, growth disorders, diabetes, neurodegenerative diseases, multiple sclerosis, Parkinson'"'"'s disease and Alzheimer'"'"'s disease.

71. The method of claim 70, wherein in the disease or condition is multiple sclerosis.

40. A pharmaceutical composition formulated for oral administration, comprising an IFN beta cytokine that contains one or more amino acid modification(s), whereby the IFN beta cytokine exhibits increased protease resistance compared to an IFN beta cytokine that does not contain the modification(s).

41. The pharmaceutical composition of claim 40, wherein the modified IFN beta cytokine has been modified by an insertion, a deletion and/or a replacement of one or more amino acid residues, whereby the cytokine is rendered resistant to proteolysis.

42. The pharmaceutical composition of claim 40, wherein:

the modified IFN beta cytokine comprises one or more amino acid replacements at one or more amino acid target positions in the unmodified cytokine.

43. The pharmaceutical composition of claim 42, wherein:

the modified IFN beta cytokine comprises one or more amino acid replacements selected from among replacement of;

M1V, M1I, M1T, M1A, M1D, M1E, M1K, M1N, M1R, M1S, M1Q, M1C, L5V, L5I, L5T, L5Q, L5H, L5A, L5D, L5E, L5K, L5R, L5N, L5S, L6D, L6E, L6K, L6N, L6Q, L6R, L6S, L6T, L6C, F8I, F8V, F8D, F8E, F8K, F8R, L9V, L9I, L9T, L9Q, L9H, L9A, L9D, L9E, L9K, L9N, L9R, L9S, Q10D, Q10E, Q10K, Q10N, Q10R, Q10S, Q10T, Q10C, R11H, R11Q, S12D, S12E, S12K, S12R, S13D, S13E, S13K, S13N, S13Q, S13R, S13T, S13C, N14D, N14E, N14K, N14Q, N14R, N14S, N14T, F15I, F15V, F15D, F15E, F15K, F15R, Q16D, Q16E, Q16K, Q16N, Q16R, Q16S, Q16T, Q16C, C17D, C17E, C17K, C17N, C17Q, C17R, C17S, C17T, C17L, K19Q, K19T, K19S, K19H, L20N, L20Q, L20R, L20S, L20T, L20D, L20E, L20K, W22S, W22H, W22D, W22E, W22K, W22R, Q23D, Q23E, Q23K, Q23R, L24D, L24E, L24K, L24R, N25H, N25S, N25Q, R27H, R27Q, L28V, L28I, L28T, L28Q, L28H, L28A, E29Q, E29H, Y30H, Y30I, L32V, L32I, L32T, L32Q, L32H, L32A, K33Q, K33T, K33S, K33H, R35H, R35Q, M36V, M36I, M36T, M36Q, M36A, D39Q, D39N, D39H, D39G, E42Q, E42N, E42H, K45Q, K45N, K45T, K45S, K45H, L47V, L47I, L47T, L47Q, L47H, L47A, K52Q, K52N, K52T, K52S, K52H, F67I, F67V, R71H, R71Q, D73Q, D73N, D73H, D73G, G78D, G78E, G78K, C78R, W79D, W79E, W79K, W79R, N80D, N80E, N80K, N80R, E81Q, E81N, E81H, T82D, T82E, T82K, T82R, I83D, I83E, I83K, I83R, I83N, I83Q, I83S, I83T, E85Q, E85H, N86D, N86E, N86K, N86R, N86Q, N86S, N86T, L87D, L87E, L87K, L87R, L87N, L87Q, L87S, L87T, A89D, A89E, A89K, A89R, N91D, N91E, N91K, N91Q, N91R, N91S, N91T, N91C, V91D, V91E, V91K, V91N, V91Q, V91R, V91S, V91T, V91C, Y92H, Y92I, Q94D, Q94E, Q94K, Q94N, Q94R, Q94S, Q94T, Q94C, I95D, I95E, I95K, I95N, I95Q, I95R, I95S, I95T, H97D, H97E, H97K, H97N, H97Q, H97R, H97S, H97T, H97C, L98D, L98E, L98K, L98N, L98Q, L98R, L98S, L98T, L98C, K99Q, K99T, K99S, K99H, V101D, V101E, V101K, V101N, V101Q, V101R, V101S, V101T, V101C, E103Q, E103H, E104Q, E104H, K105Q, K105T, K105S, K105H, E107Q, E107N, E107H, K108Q, K108N, K108T, K108S, K108H, E109Q, E109N, E109H, D110Q, D110N, D110H, D110G, F111I, F111V, R113H, R113Q, L116V, L116I, L116T, L116Q, L116H, L116A, L120V, L120I, L120T, L120Q, L120H, L120A, K123Q, K123N, K123T, K123S, K123H, R124H, R124Q, R128H, R128Q, L131V, L130I, L130T, L130Q, L130H, L130A, K134Q, K134N, K134T, K134S, K134H, K136Q, K136N, K136T, K136S, K136H, E137Q, E137N, E137H, Y138H, Y138I, R152H, R152Q, Y155H, Y155I, R159H, R159Q, Y163H, Y163I, R165H and R165Q; and

the one or more amino acid replacements occur in a mature IFN beta cytokine having the sequence set forth in SEQ ID NO;

196 or in a sequence-related IFN beta cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

96.

44. The pharmaceutical composition of claim 43, wherein:

the modified IFN beta cytokine comprises one or more amino acid replacements selected from among replacement of;

M1V, M1I, M1T, M1A, M1D, M1E, M1K, M1N, M1R, M1S, M1Q, L5V, L5I, L6K, L6N, L6Q, L6R, L6C, F8I, F8V, L9V, L9I, L9T, L9Q, L9H, L9A, L9S, Q10S, Q10T, R11H, R11Q, S13Q, N14D, N14E, N14K, N14Q, N14R, N14S, N14T, F15I, F15V, Q16N, Q16R, Q16S, Q16T, C17K, C17Q, C17R, K19T, K19S, K19H, L20N, L20Q, L20R, L20S, L20T, Q23R, N25H, N25S, N25Q, R27H, R27Q, L28V, L28I, E29Q, Y30I, L32V, L32I, K33T, K33S, K33H, R35H, R35Q, M36V, M36I, D39H, D39G, K45T, K45S, K45H, K52T, K52S, K52H, D73H, D73G, I83Q, I83S, I83T, E85H, N86D, N86R, N86Q, N86S, N86T, L87Q, L87S, L87T, N91Q, N91S, N91T, V91Q, V91S, V91T, Y92I, Q94S, Q94T, I95K, I95N, I95Q, I95R, I95S, I95T, H97N, H97Q, H97R, H97S, H97T, L98D, L98E, L98K, L98N, L98Q, L98R, L98C, K99T, K99S, K99H, V101D, V101E, V101K, V101N, V101Q, V101R, V101S, V101T, V101C, E103H, K105T, K105S, K105H, K108T, K108S, K108H, D110H, D110G, K123T, K123S, K123H, K134T, K134S, K134H, K136T, K136S, K136H, Y138H, Y138I, R152H, R152Q, Y155H, Y155I, R159H and R159Q; and

the one or more amino acid replacements occur in a mature IFN beta cytokine having the sequence set forth in SEQ ID NO;

196 or in a sequence-related IFN beta cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

196.

45. The pharmaceutical composition of claim 43, wherein:

the modified IFN beta cytokine comprises one or more amino acid replacements selected from among replacement of;

D39N, E42N, E42H, K45N, L47V, L47I, K52N, F67I, F67V, R71H, R71Q, E81H, E107N, E107H, K108N, E109N, E109H, F111I, F111V, R113H, L116V, L116I, L121V, L120I, K123N, R124H, R124Q, R128H, R128Q, L131V, L130I, K134N, K136N, E137H, Y163H, Y163, R165H and R165Q; and

the one or more amino acid replacements occur in a mature IFN beta cytokine having the sequence set forth in SEQ ID NO;

196 or in a sequence-related IFN beta cytokine at corresponding amino acid position(s) relative to SEQ ID NO;

196.

46. The pharmaceutical composition of claim 43, wherein the IFN beta cytokine comprises the sequence of amine acids set forth in any of SEQ ID NOS:

233-289 or 989-1302.

47. The pharmaceutical composition of claim 43, wherein the IFN beta cytokine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the amino acid replacements in its sequence of amino acid residues.

48. The pharmaceutical composition of claim 43, wherein the IFN beta cytokine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid replacements in its sequence of amino acid residues.

49. The pharmaceutical composition of claim 40, further comprising a pharmaceutically acceptable carrier or excipient.

50. The pharmaceutical composition of claim 49, wherein the pharmaceutically acceptable carrier or excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant, and a wetting agent.

51. The pharmaceutical composition of claim 40, further comprising a pharmaceutically acceptable additive.

52. The pharmaceutical composition of claim 51, wherein the pharmaceutically acceptable additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle and a preservative.

53. The pharmaceutical composition of claim 40, wherein the composition is in the form of a liquid, a solution, a suspension, an aerosol, a tablet, a lozenge or a capsule.

54. The pharmaceutical composition of claim 53, wherein the composition is in the form of a tablet or a capsule.

55. The pharmaceutical composition of claim 40, wherein the pharmaceutical composition is formulated for controlled-release of the IFN beta cytokine.

56. The pharmaceutical composition of claim 40, wherein the IFN beta cytokine has been modified by removing proteolytic digestion sites in the IFN beta cytokine.

57. The pharmaceutical composition of claim 40, wherein the IFN beta cytokine has an increased half-life compared to the unmodified IFN beta cytokine.

58. The pharmaceutical composition of claim 40, wherein the IFN beta cytokine exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract.

59. The pharmaceutical composition of claim 40, wherein the IFN beta cytokine exhibits increased antiviral and/or anti-proliferative activity compared to the unmodified IFN beta cytokine.

60. The pharmaceutical composition of claim 40, wherein the IFN beta cytokine exhibits comparable antiviral and/or anti-proliferative activity compared to the unmodified IFN beta cytokine.

61. The pharmaceutical composition of claim 40, wherein the IFN beta cytokine exhibits decreased antiviral and/or anti-proliferative activity compared to the unmodified IFN beta cytokine.

66. A method, comprising treating a subject by orally administering the pharmaceutical composition of claim 40, wherein the subject has a disease or condition that is treated by administration of an IFN beta cytokine.

67. The method of claim 66, wherein in the disease or condition is selected from among infectious diseases, allergies, microbial diseases, pregnancy related diseases, bacterial diseases, heart diseases, viral diseases, histological diseases, genetic diseases, blood related diseases, fungal diseases, adrenal diseases, cancers, liver diseases, autoimmune diseases, growth disorders, diabetes, neurodegenerative diseases, multiple sclerosis, Parkinson'"'"'s disease and Alzheimer'"'"'s disease.

68. The method of claim 67, wherein in the disease or condition is multiple sclerosis.