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

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.

1 . A modified growth hormone, comprising one or more amino acid replacements in its sequence of amino acid residues, wherein:
 the modified growth hormone exhibits increased resistance to proteolysis compared to the unmodified growth hormone 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: P59S, E66N, E66H, E88Q, E88N, E88H, F92I, F92V, R94H, R94Q, E129Q, E129N, E129H, D130Q, D130N, P133S, P133A, R134H, R134Q, K140Q, Y143H, Y143I, K145Q, K145N, F146I, F146V, D147Q, D147N, R183Q; and   the one or more amino acid replacements occur in a mature growth hormone having the sequence set forth in SEQ ID NO: 216 or in a sequence-related growth hormone at corresponding amino acid position(s) relative to SEQ ID NO: 216   
     
     
         2 . The modified growth hormone of  claim 1 , wherein the unmodified growth hormone contains the amino acids residues having the sequence set forth in SEQ ID NO: 216. 
     
     
         3 . The growth hormone 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. 
     
     
         4 . The growth hormone 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. 
     
     
         5 . The growth hormone 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: E56Q, E56N, E56H, P59S, P59A, R64H, R64Q, E65Q, E65N, E65H, E66Q, E66N, E66H, E88Q, E88N, E88H, F921, F92V, R94H, R94Q, L101V, L101I, E129Q, E129N, E129H, D130Q, D130N, P133S, P133A, R134H, R134Q, K140Q, K140N, Y143H, Y143I, K145Q, K145N, F146I, F146V, D147Q, D147N, R183H, R183Q, E186Q, E186N and E186H; and   the one or more amino acid replacements occur in a mature growth hormone having the sequence set forth in SEQ ID NO: 216 or in a sequence-related growth hormone at corresponding amino acid position(s) relative to SEQ ID NO: 216.   
     
     
         6 . The growth hormone of  claim 1 , wherein only the primary amino acid sequence is modified, and the growth hormone exhibits increased resistance to proteolysis. 
     
     
         7 . A growth hormone of  claim 1 , wherein the cytokine comprises the sequence of amino acids set forth in any of SEQ ID NOS: 853, 861, 862, 863, 864, 865, 866, 867, 868, 869, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 883, 884, 885, 886, 887, 888, 889, 890 or 892. 
     
     
         8 . The growth hormone of  claim 1  that exhibits increased stability compared to the unmodified growth hormone. 
     
     
         9 . The growth hormone of  claim 8 , wherein the growth hormone exhibits increased stability to proteases, human blood lysate or human serum. 
     
     
         10 . The growth hormone of  claim 1  that exhibits increased protein half-life in vitro or in vivo compared to the unmodified growth hormone. 
     
     
         11 . The growth hormone of  claim 1  that exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract. 
     
     
         12 . The growth hormone of  claim 1  that exhibits increased resistance to proteolysis by a protease in the serum. 
     
     
         13 . The growth hormone of  claim 1 , wherein increased resistance to proteolysis is due to replacement of one or more amino acids at target positions in an unmodified growth hormone that increase resistance of the growth hormone to digestion by a protease. 
     
     
         14 . The growth hormone of  claim 1  that exhibits increased biological activity compared to the unmodified growth hormone. 
     
     
         15 . The growth hormone of  claim 1  that exhibits decreased biological activity compared to the unmodified growth hormone. 
     
     
         16 . A nucleic acid molecule encoding a modified growth hormone of  claim 1 . 
     
     
         17 . A vector, comprising a nucleic acid molecule of  claim 16 . 
     
     
         18 . A eukaryotic cell, comprising the nucleic acid molecule of  claim 16 . 
     
     
         19 . A eukaryotic cell, comprising the vector of  claim 17 . 
     
     
         20 . A collection of nucleic acid molecules, comprising a plurality of the molecules of  claim 16 . 
     
     
         21 . A collection of nucleic acid molecules, comprising a plurality of the vectors of  claim 17 . 
     
     
         22 . A method for expression of a modified growth hormone, comprising:
 introducing a nucleic acid of  claim 16  into a host; and   culturing the cell under conditions, whereby the encoded modified growth hormone is expressed.   
     
     
         23 . The method of  claim 22 , further comprising isolating the modified growth hormone. 
     
     
         24 . The method of  claim 22 , wherein the host cell is a eukaryotic host cell or a bacterial cell. 
     
     
         25 . A pharmaceutical composition, comprising a growth hormone of  claim 1 . 
     
     
         26 . The pharmaceutical composition of  claim 25 , further comprising a pharmaceutically acceptable carrier or excipient. 
     
     
         27 . The pharmaceutical composition of  claim 26 , wherein the pharmaceutically acceptable carrier or excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant and a wetting agent. 
     
     
         28 . The pharmaceutical composition of  claim 25 , further comprising a pharmaceutically acceptable additive. 
     
     
         29 . The pharmaceutical composition of  claim 28 , wherein the pharmaceutically acceptable additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle and a preservative. 
     
     
         30 . The pharmaceutical composition of  claim 25 , wherein the composition is in the form of a liquid, a solution, a suspension, an aerosol, a tablet, a lozenge or a capsule. 
     
     
         31 . The pharmaceutical composition of  claim 25 , formulated for oral, parenteral, intravenous, intradermal, subcutaneous, buccal, inhalation, intramuscular, rectal or topical administration. 
     
     
         32 . The pharmaceutical composition of  claim 31 , formulated for oral administration. 
     
     
         33 . The pharmaceutical composition of  claim 32 , wherein the pharmaceutical composition is formulated for oral administration to the mouth or gastrointestinal tract. 
     
     
         34 . The pharmaceutical composition of  claim 25 , wherein the pharmaceutical composition is formulated for controlled-release of the growth hormone. 
     
     
         35 . A pharmaceutical composition formulated for oral administration, comprising a growth hormone that contains one or more amino acid modification(s), whereby the growth hormone exhibits increased protease resistance compared to a growth hormone that does not contain the modification(s). 
     
     
         36 . The pharmaceutical composition of  claim 35 , wherein the modified growth hormone has been modified by a insertion, a deletion and/or a replacement of one or more amino acid residues, whereby the cytokine is rendered resistant to proteolysis. 
     
     
         37 . The pharmaceutical composition of  claim 35 , wherein:
 the modified growth hormone comprises one or more amino acid replacements at one or more amino acid target positions in the unmodified cytokine.   
     
     
         38 . The pharmaceutical composition of  claim 37 , wherein:
 the modified growth hormone comprises one or more amino acid replacements selected from among replacement of: E56Q, E56N, E56H, P59S, P59A, R64H, R64Q, E65Q, E65N, E65H, E66Q, E66N, E66H, E88Q, E88N, E88H, F92I, F92V, R94H, R94Q, L101V, L101I, E129Q, E129N, E129H, D130Q, D130N, P133S, P133A, R134H, R134Q, K140Q, K140N, Y143H, Y143I, K145Q, K145N, F146I, F146V, D147Q, D147N, R183H, R183Q, E186Q, E186N and E186H; and   the one or more amino acid replacements occur in a mature growth hormone having the sequence set forth in SEQ ID NO: 216 or in a sequence-related growth hormone at corresponding amino acid position(s) relative to SEQ ID NO: 216.   
     
     
         39 . The pharmaceutical composition of  claim 35 , further comprising a pharmaceutically acceptable carrier or excipient. 
     
     
         40 . The pharmaceutical composition of  claim 39 , wherein the pharmaceutically acceptable carrier or excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant and a wetting agent. 
     
     
         41 . The pharmaceutical composition of  claim 35 , further comprising a pharmaceutically acceptable additive. 
     
     
         42 . The pharmaceutical composition of  claim 41 , wherein the pharmaceutically acceptable additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle and a preservative. 
     
     
         43 . The pharmaceutical composition of  claim 35 , wherein the composition is in the form of a liquid, a solution, a suspension, an aerosol, a tablet, a lozenge or a capsule. 
     
     
         44 . The pharmaceutical composition of  claim 35 , wherein the pharmaceutical composition is formulated for controlled-release of the growth hormone. 
     
     
         45 . The pharmaceutical composition of  claim 35 , wherein the growth hormone has been modified by removing proteolytic digestion sites in the growth hormone. 
     
     
         46 . The pharmaceutical composition of  claim 35 , wherein the growth hormone has an increased half-life compared to the unmodified growth hormone. 
     
     
         47 . The pharmaceutical composition of  claim 35 , wherein the growth hormone exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract. 
     
     
         48 . The pharmaceutical composition of  claim 35 , wherein the growth hormone exhibits increased biological activity compared to the unmodified growth hormone. 
     
     
         49 . The pharmaceutical composition of  claim 35 , wherein the growth hormone exhibits decreased biological activity compared to the unmodified growth hormone. 
     
     
         50 . A pharmaceutical composition, comprising a nucleic acid molecule of  claim 16 . 
     
     
         51 . A method, comprising treating a subject by administering the pharmaceutical composition of  claim 25 , wherein the subject has a disease or condition that is treated by administration of a growth hormone. 
     
     
         52 . The method of  claim 51 , wherein in the disease or condition is selected from among growth hormone deficiency and acromegaly. 
     
     
         53 . A method, comprising treating a subject by orally administering the pharmaceutical composition of  claim 35 , wherein the subject has a disease or condition that is treated by administration of a growth hormone. 
     
     
         54 . The method of  claim 53 , wherein in the disease or condition is selected from among growth hormone deficiency and acromegaly. 
     
     
         55 . A method, comprising treating a subject by administering the pharmaceutical composition of  claim 50 , wherein the subject has a disease or condition that is treated by administration of a growth hormone. 
     
     
         56 . The method of  claim 55 , wherein in the disease or condition is selected from among growth hormone deficiency and acromegaly.