Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
First Claim
Patent Images
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.
4 Assignments
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Accused Products
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.
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Citations
71 Claims
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1. A modified interferon beta (IFN beta) cytokine, comprising one or more amino acid replacements in its sequence of amino acid residues, wherein:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 63, 64, 65)
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19. A modified interferon beta (IFN beta) cytokine, comprising two or more amino acid replacements in its sequence of amino acid residues, wherein:
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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, 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. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 62, 69, 70, 71)
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- 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).
Specification