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 pharmaceutical composition, comprising a modified cytokine, wherein: 
 the modified cytokine comprises a sequence of amino acids set forth in any of SEQ ID Nos. 2-181, 233-1303 or a structural homolog thereof;    the modified cytokine is more resistant to proteolysis compared to a wild-type cytokine; and    the composition is formulated for oral delivery.    
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein the cytokine is selected from among interleukin-10 (IL-10), interferon beta (IFN-β), interferon alpha-2a (IFN α-2a), interferon alpha-2b (IFN α-2b), interferon gamma (IFN-γ), granulocyte colony stimulating factor (G-CSF), leukemia inhibitory factor (LIF), human growth hormone (hGH), ciliary neurotrophic factor (CNTF), leptin, oncostatin M, interleukin-6 (IL-6) and interleukin-12 (IL-12), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), Flt3 ligand and stem cell factor (SCF).  
     
     
         3 . The pharmaceutical composition of  claim 1 , wherein the modified cytokine is selected from among modified cytokines comprising amino acids set forth in any of SEQ ID Nos. 2-181, 233-1303 or a structural homolog thereof.  
     
     
         4 . The pharmaceutical composition of  claim 1 , wherein the modified cytokine is more resistant to proteolysis by a protease of the luminal gastrointestinal tract.  
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the cytokine is selected from among interleukin-10 (IL-10), interferon beta (IFN-β), interferon alpha-2a (IFN α-2a), interferon alpha-2b (IFN α-2b), interferon α-2c (IFN α-2c), interferon gamma (IFN-γ), granulocyte colony stimulating factor (G-CSF), leukemia inhibitory factor (LIF), human growth hormone (hGH), ciliary neurotrophic factor (CNTF), leptin, oncostatin M, interleukin-6 (IL-6) and interleukin-12 (IL-12), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-13 (IL-13), Flt3 ligand and stem cell factor (SCF).  
     
     
         6 . The pharmaceutical composition of  claim 1 , further comprising a pharmaceutically acceptable excipient.  
     
     
         7 . The pharmaceutical composition of  claim 6 , wherein the excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant and a wetting agent.  
     
     
         8 . The pharmaceutical composition of  claim 1 , further comprising a pharmaceutically-acceptable additive.  
     
     
         9 . The pharmaceutical composition of  claim 8 , wherein the additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle and a preservative.  
     
     
         10 . The pharmaceutical composition of  claim 6  that is formulated as a tablet.  
     
     
         11 . The pharmaceutical composition of  claim 6  that is formulated as a capsule.  
     
     
         12 . The pharmaceutical composition of  claim 6  that is formulated as a liquid.  
     
     
         13 . The pharmaceutical composition of  claim 6  that is formulated as a lozenge.  
     
     
         14 . The pharmaceutical composition of  claim 13  that is formulated for controlled-release.  
     
     
         15 . The pharmaceutical composition of  claim 1 , formulated for oral administration to the mouth or gastrointestinal tract.  
     
     
         16 . The pharmaceutical composition of  claim 1 , wherein the cytokine has been modified by removing proteolytic digestion sites in the cytokine thereby increasing the overall stability of the cytokine structure.  
     
     
         17 . The pharmaceutical composition of  claim 16 , wherein stability is assessed by measuring residual biological activity.  
     
     
         18 . The pharmaceutical composition of  claim 1 , wherein the modified cytokine has an increased half-life compared to an unmodified or wild-type cytokine.  
     
     
         19 . The pharmaceutical composition of  claim 1 , wherein the modified cytokine comprises one or more amino acid replacements at one or more target positions.  
     
     
         20 . The pharmaceutical composition of  claim 19 , 
 wherein the modified cytokine is a human interferon, and    the one or more target positions are selected from among positions 41, 58, 78, 107, 117, 125, 133 and 159 of SEQ ID NO: 182, positions 41, 59, 79, 108, 118, 126, 134 and 160 of SEQ ID NO: 183, positions 27, 33, 41, 58, 78, 89, 107, 109, 110, 111, 113, 117, 121, 125, 133 and 159 of SEQ ID NO: 185, positions 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134 and 160 of SEQ ID NOS: 186-195, positions 39, 42, 45, 47, 52, 67, 71, 73, 81, 107, 108, 109, 110, 111, 113, 116, 120, 123, 124, 128, 130, 134, 136, 137, 163 and 165 of SEQ ID NO: 196, and positions 33, 37, 40, 41, 42, 58, 61, 64, 65 and 66 of SEQ ID NO: 199.    
     
     
         21 . The pharmaceutical composition of  claim 19 , 
 wherein the modified cytokine comprises one or more mutations at one or more target positions in a cytokine,    wherein the one or more mutations comprises an insertion, a deletion and/or a replacement of one or more native amino acid residues, and    wherein the one or more target positions are selected from among positions 41, 58, 78, 107, 117, 125, 133 and 159 of SEQ ID NO: 182, positions 41, 59, 79, 108, 118, 126, 134 and 160 of SEQ ID NO: 183, positions 27, 33, 41, 58, 78, 89, 107, 109, 110, 111, 113, 117, 121, 125, 133 and 159 of SEQ ID NO: 185, positions 27, 33, 41, 59, 79, 90, 108, 110, 111, 112, 114, 118, 122, 126, 134 and 1600 of SEQ ID NOS: 186-195 positions 39, 42, 45, 47, 52, 67, 71, 73, 81, 107, 108, 109, 110, 111, 113, 116, 120, 123, 124, 128, 130, 134, 136, 137, 163 and 165 of SEQ ID NO: 196, and positions 33, 37, 40, 41, 42, 58, 61, 64, 65 and 66 of SEQ ID NO: 199, positions 49, 50, 52, 53, 54, 55, 56, 57, 59, 60, 67, 68, 71, 72, 74, 75, 78, 81, 84, 85, 86, and 88 of SEQ ID NO: 200, positions 43, 45, 48, 49, 52, 53, 55, 72, 75, 76, 123, 129, 130, 131, 162, and 165 of SEQ ID NO: 201, positions 38, 41, 45, 46, 48, 49, 51, 60, 63, 67, 92, 93, 119, 120, 123, and 124 of SEQ ID NO: 202, positions 3, 40, 42, 43, 55, 58, 59, 61, 89, 90, 91, 95, and 96 of SEQ ID NO: 203, positions 43, 45, 48, 49, 52, 53, 60, 61, 65, 67, 68, 72, 100, 103, 104, 106, 107, 109, 110, and 132 of SEQ ID NO: 204, positions 37, 43, 46, 59, 63, 66, 96, 100, 101, and 103 of SEQ ID NO: 205, positions 27, 31, 34, 37, 54, 58, 61, 62, 63, 96, 98, 99, 100, 102, 103, 106, 107, 108, 109, 134, and 137 of SEQ ID NO: 206, positions 26, 37, 53, 60, 61, 64, 66, 100, 102, 103, and 126 of SEQ ID NO: 207, positions 32, 34, 39, 46, 47, 56, 84, 85, 88, 89, 90, 102, 110, and 111 of SEQ ID NO: 208, positions 32, 34, 38, 48, 79, 82, 85, 86, 88, 107, 108, 110, and 111 of SEQ ID NO: 209, positions 62, 64, 66, 67, 86, 89, 92, 100, 102, 104, 131, 132, 133, 135, 136, 138, 140, 143, 148, and 151 of SEQ ID NO: 212, positions 69, 70, 85, 99, 102, 104, 106, 109, 137, 143, 146, 148, 149, 153, 154, and 156 of to SEQ ID NO: 213, positions 59, 60, 63, 65, 84, 87, 89, 91, 94, 97, 99, 100, 103, and 106 of SEQ ID NO: 214, positions 56, 61, 66, 67, 68, 70, 72, 75, 78, 79, 82, 89, 92, 93, 107, 110, 111, 115, 117, 124, 125, 127, 128, 129, and 189 of SEQ ID NO: 215, positions 56, 59, 64, 65, 66, 88, 92, 94, 101, 129, 130, 133, 134, 140, 143, 145, 146, 147, 183, and 186 of SEQ ID NO: 216, and positions 64, 65, 66, 68, 69, 75, 77, 92, 98, 103, 105, 108, 133, 138, 139, 140, 149, 156, 178, and 181 of SEQ ID NO: 217.    
     
     
         22 . A method, comprising treating a subject by administering the pharmaceutical composition of  claim 1 , where in the subject has a disease or condition that is treated by administration of a cytokine.  
     
     
         23 . The method of  claim 22 , wherein the subject has a disease or condition that is selected from among an infectious disease, an allergic disease, asthma, a microbial disease, a pregnancy-related disease, a bacterial disease, a heart disease, a viral disease, a histological disease, a genetic disease, a blood-related disease, a fungal disease, an adrenal disease, a cancer, a liver disease, an autoimmune disease, a growth disorder, diabetes, a neurodegenerative disease, multiple sclerosis, Parkinson's disease, Alzheimer's disease, a fibrosis, a liver disease or condition, a degenerative disease, obesity, tissue damage, a hematopoietic-associated disease, a sclerosis and an inflammatory disease.  
     
     
         24 . The method of  claim 22 , wherein the modified cytokine is an IFN-β.  
     
     
         25 . The method of  claim 24 , wherein the subject has multiple sclerosis.  
     
     
         26 . The method of  claim 22 , wherein the modified cytokine is an IFNα.  
     
     
         27 . The method of  claim 26 , wherein the subject has a disease or condition selected from among type B chronic hepatitis, type C chronic hepatitis, a herpes infection, a melanoma, a Kaposi sarcoma, and a lymphoma.  
     
     
         28 . The method of  claim 22 , wherein the modified cytokine is an IL-10.  
     
     
         29 . The method of  claim 28 , wherein the subject has a disease or condition selected from among the disease or condition is selected from among an inflammatory disease or condition selected from chronic liver injury, chronic liver disease and a myeloma.  
     
     
         30 . The method of  claim 22 , wherein the modified cytokine is IFN-γ.  
     
     
         31 . The method of  claim 30 , wherein the subject has a disease or condition selected from among interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis and adjunctive immunotherapy for immunosuppressed patients.  
     
     
         32 . The method of  claim 22 , wherein the modified cytokine is a G-CSF.  
     
     
         33 . The method of  claim 32 , wherein the subject has a disease or condition selected from among Crohn's disease, a cardiac disease, an acquired congenital neutropenia, a congenital neutropenia and asthma.  
     
     
         34 . The method of  claim 22 , wherein the modified cytokine is an LIF.  
     
     
         35 . The method of  claim 34 , wherein the subject has a disease or condition selected from among myocardial infarction, multiple sclerosis, olfactory epithelium replacement stimulation and axonal atrophy.  
     
     
         36 . The method of  claim 22 , wherein the modified cytokine is an hGH.  
     
     
         37 . The method of  claim 36 , wherein the subject has a disease or condition selected from among growth hormone deficiency and acromegaly.  
     
     
         38 . The method of  claim 22 , wherein the modified cytokine is a CNTF.  
     
     
         39 . The method of  claim 38 , wherein subject has a disease or condition selected from among retinal degeneration, a neurodegenerative disease and an auditory degenerative disease.  
     
     
         40 . The method of  claim 39 , wherein the neurodegenerative disease is Huntingdon's disease.  
     
     
         41 . The method of  claim 22 , wherein the modified cytokine is a leptin.  
     
     
         42 . The method of  claim 41 , wherein the subject has a disease or condition selected from among obesity, pancreatitis and endometriosis.  
     
     
         43 . The method of  claim 22 , wherein the modified cytokine is oncostatin M.  
     
     
         44 . The method of  claim 43 , wherein the subject has a disease or condition selected from among a chronic inflammatory disease, rheumatoid arthritis, multiple sclerosis and tissue damage.  
     
     
         45 . The method of  claim 22 , wherein the modified cytokine is an IL-6.  
     
     
         46 . The method of  claim 45 , wherein the subject has a disease or condition selected from among liver injury, Crohn's disease and a hematopoietic-associated disease.  
     
     
         47 . The method of  claim 22 , wherein the modified cytokine is an IL-12.  
     
     
         48 . The method of  claim 47 , wherein the subject has a disease or condition selected from among coxsackievirus, a neuroblastoma, a melanoma, renal cell carcinoma, and mucosal immunity induction.  
     
     
         49 . The method of  claim 22 , wherein the modified cytokine is an erythropoietin (EPO).  
     
     
         50 . The method of  claim 49 , wherein the subject has a disease or condition selected from among hypoxia, myocardial ischemia, anemia with renal failure and a cancer.  
     
     
         51 . The method of  claim 22 , wherein the modified cytokine is a GM-CSF.  
     
     
         52 . The method of  claim 51 , wherein the subject has a disease or condition selected from among leukemia, a melanoma, a breast carcinoma, a liver carcinoma, a renal cell carcinoma, an autoimmune disease and adjunctive therapy for immunosuppressed patients.  
     
     
         53 . The method of  claim 22 , wherein the modified cytokine is an IL-2.  
     
     
         54 . The method of  claim 53 , wherein the subject has a disease or condition selected from among a melanoma or a colon carcinoma.  
     
     
         55 . The method of  claim 54 , wherein the immune system is reactivated after chemotherapy.  
     
     
         56 . The method of  claim 22 , wherein the modified cytokine is an IL-3.  
     
     
         57 . The method of  claim 56 , wherein the subject has a disease or condition selected from among a leukemia, a motor neuropathy, amyotrophic lateral sclerosis and asthma.  
     
     
         58 . The method of  claim 22 , wherein the modified cytokine is an IL-4.  
     
     
         59 . The method of  claim 58 , wherein the subject has a disease or condition is selected from among allergic asthma and lupus.  
     
     
         60 . The method of  claim 22 , wherein the modified cytokine is an IL-5.  
     
     
         61 . The method of  claim 60 , wherein the subject has a condition selected from among a parasite, asthma, and an allergic disease accompanied by eosinophilia.  
     
     
         62 . The method of  claim 22 , wherein the modified cytokine is an IL-13.  
     
     
         63 . The method of  claim 62 , wherein the subject has a disease or condition selected from among an intracellular infection, a B-cell cancer and asthma.  
     
     
         64 . The method of  claim 22 , wherein the modified cytokine is a Flt3 ligand.  
     
     
         65 . The method of  claim 64 , wherein the subject has a disease or condition selected from among prostate cancer, myeloid leukemia and engraftment of allogenic hematopoietic stem cells.  
     
     
         66 . The method of  claim 22 , wherein the modified cytokine is a SCF.  
     
     
         67 . The method of  claim 66 , wherein the subject has a disease or condition selected from among a hepatic injury, asthma and hematopoietic engraftment.