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 erythropoietin cytokine, comprising one or more amino acid replacements in its sequence of amino acid residues, wherein: 
 the modified erythropoietin cytokine exhibits increased resistance to proteolysis compared to the unmodified erythropoietin 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: D by Q at position 43; K by Q at position 45; K by N at position 45; F by V at position 48; Y by H at position 49; Y by I at position 49; K by N at position 52; R by H at position 53; R by Q at position 53; E by Q at position 55; E by N at position 55; E by H at position 55; E by Q at position 72; E by N at position 72; E by H at position 72; L by V at position 75; L by I at position 75; R by H at position 76; P by S at position 121; P by A at position 121; P by S at position 122; P by A at position 122; D by Q at position 123; D by N at position 123; P by S at position 129; P by A at position 129; L by V at position 130; L by I at position 130; R by H at position 131; R by Q at position 131; R by H at position 162; R by Q at position 162; D by Q at position 165; and D by N at position 165; and    the one or more amino acid replacements occur in a mature erythropoietin cytokine having the sequence set forth in SEQ ID NO: 201 or in a sequence-related erythropoietin cytokine at corresponding amino acid position(s) relative to SEQ ID NO: 201.    
     
     
         2 . The modified erythropoietin cytokine of  claim 1 , wherein the unmodified erythropoietin cytokine contains the amino acids residues having the sequence set forth in SEQ ID NO: 201.  
     
     
         3 . The erythropoietin 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.  
     
     
         4 . The erythropoietin 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.  
     
     
         5 . The erythropoietin 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: D by Q at position 43; D by N at position 43; K by Q at position 45; K by N at position 45; F by I at position 48; F by V at position 48; Y by H at position 49; Y by I at position 49; K by Q at position 52; K by N at position 52; R by H at position 53; R by Q at position 53; E by Q at position 55; E by N at position 55; E by H at position 55; E by Q at position 72; E by N at position 72; E by H at position 72; L by V at position 75; L by I at position 75; R by H at position 76; R by Q at position 76; P by S at position 121; P by A at position 121; P by S at position 122; P by A at position 122; D by Q at position 123; D by N at position 123; P by S at position 129; P by A at position 129; L by V at position 130; L by I at position 130; R by H at position 131; R by Q at position 131; R by H at position 162; R by Q at position 162; D by Q at position 165; and D by N at position 165; and    the one or more amino acid replacements occur in a mature erythropoietin cytokine having the sequence set forth in SEQ ID NO: 201 or in a sequence-related erythropoietin cytokine at corresponding amino acid position(s) relative to SEQ ID NO: 201.    
     
     
         6 . The erythropoietin cytokine of  claim 1 , wherein only the primary amino acid sequence is modified, and the erythropoietin cytokine exhibits increased resistance to proteolysis.  
     
     
         7 . An erythropoietin cytokine of  claim 1 , wherein the cytokine comprises the sequence of amino acids set forth in any of SEQ ID NOS: 940, 942, 943, 945, 946, 947, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, and 977.  
     
     
         8 . The erythropoietin cytokine of  claim 1  that exhibits increased stability compared to the unmodified erythropoietin cytokine.  
     
     
         9 . The erythropoietin cytokine of  claim 8 , wherein the erythropoietin cytokine exhibits increased stability to proteases, human blood lysate or human serum.  
     
     
         10 . The erythropoietin cytokine of  claim 1  that exhibits increased protein half-life in vitro or in vivo compared to the unmodified erythropoietin cytokine.  
     
     
         11 . The erythropoietin cytokine of  claim 1  that exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract.  
     
     
         12 . The erythropoietin cytokine of  claim 1  that exhibits increased resistance to proteolysis by a protease in the serum.  
     
     
         13 . The erythropoietin 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 erythropoietin cytokine that increase resistance of the erythropoietin cytokine to digestion by a protease.  
     
     
         14 . The erythropoietin cytokine of  claim 1  that exhibits increased biological activity compared to the unmodified erythropoietin cytokine.  
     
     
         15 . The erythropoietin cytokine of  claim 1  that exhibits decreased biological activity compared to the unmodified erythropoietin cytokine.  
     
     
         16 . A nucleic acid molecule encoding an erythropoietin cytokine 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 an erythropoietin cytokine, comprising: 
 introducing a nucleic acid of  claim 16  into a host cell; and    culturing the cell under conditions, whereby the encoded erythropoietin cytokine is expressed.    
     
     
         23 . The method of  claim 22 , further comprising isolating the erythropoietin cytokine.  
     
     
         24 . The method of  claim 22 , wherein the host cell is a eukaryotic cell or a bacterial cell.  
     
     
         25 . A pharmaceutical composition, comprising an erythropoietin cytokine 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 erythropoietin cytokine.  
     
     
         35 . A pharmaceutical composition formulated for oral administration, comprising an erythropoietin cytokine that contains one or more amino acid modification(s), whereby the erythropoietin cytokine exhibits increased protease resistance compared to an erythropoietin cytokine that does not contain the modification(s).  
     
     
         36 . The pharmaceutical composition of  claim 35 , wherein the modified erythropoietin 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.  
     
     
         37 . The pharmaceutical composition of  claim 35 , wherein: 
 the modified erythropoietin cytokine 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 erythropoietin cytokine comprises one or more amino acid replacements selected from among replacement of: D by Q at position 43; D by N at position 43; K by Q at position 45; K by N at position 45; F by I at position 48; F by V at position 48; Y by H at position 49; Y by I at position 49; K by Q at position 52; K by N at position 52; R by H at position 53; R by Q at position 53; E by Q at position 55; E by N at position 55; E by H at position 55; E by Q at position 72; E by N at position 72; E by H at position 72; L by V at position 75; L by I at position 75; R by H at position 76; R by Q at position 76; P by S at position 121; P by A at position 121; P by S at position 122; P by A at position 122; D by Q at position 123; D by N at position 123; P by S at position 129; P by A at position 129; L by V at position 130; L by I at position 130; R by H at position 131; R by Q at position 131; R by H at position 162; R by Q at position 162; D by Q at position 165; and D by N at position 165; and    the one or more amino acid replacements occur in a mature erythropoietin cytokine having the sequence set forth in SEQ ID NO: 201 or in a sequence-related erythropoietin cytokine at corresponding amino acid position(s) relative to SEQ ID NO: 201.    
     
     
         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 erythropoietin cytokine.  
     
     
         45 . The pharmaceutical composition of  claim 35 , wherein the erythropoietin cytokine has been modified by removing proteolytic digestion sites in the erythropoietin cytokine.  
     
     
         46 . The pharmaceutical composition of  claim 35 , wherein the erythropoietin cytokine has an increased half-life compared to the unmodified erythropoietin cytokine.  
     
     
         47 . The pharmaceutical composition of  claim 35 , wherein the erythropoietin cytokine exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract.  
     
     
         48 . The pharmaceutical composition of  claim 35 , wherein the erythropoietin cytokine exhibits increased biological activity compared to the unmodified erythropoietin cytokine.  
     
     
         49 . The pharmaceutical composition of  claim 35 , wherein the erythropoietin cytokine exhibits decreased biological activity compared to the unmodified erythropoietin cytokine.  
     
     
         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 an erythropoietin cytokine.  
     
     
         52 . The method of  claim 51 , wherein in the disease or condition is selected from among hypoxia, myocardial ischemia, or anemia associated with renal failure, or cancer.  
     
     
         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 an erythropoietin cytokine.  
     
     
         54 . The method of  claim 53 , wherein in the disease or condition is selected from among hypoxia, myocardial ischemia, or anemia associated with renal failure, or cancer.  
     
     
         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 an erythropoietin cytokine.  
     
     
         56 . The method of  claim 55 , wherein in the disease or condition is selected from among hypoxia, myocardial ischemia, or anemia associated with renal failure, or cancer.