Rational evolution of cytokines for higher stability, the cytokines and encoding nucleic acid molecules
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 granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine, comprising one or more amino acid replacements in its sequence of amino acid residues, wherein:
the modified GM-CSF cytokine exhibits increased resistance to proteolysis by a serum protease or a protease of the digestive tract compared to the unmodified GM-CSF cytokine that does not comprise the one or more amino acid replacements.
2 . The modified GM-CSF cytokine of claim 1 , wherein:
the one or more amino acid replacements and positions thereof are selected from among replacement of: E38Q, E38N, E38H, E41Q, E41N, E41H, E45Q, E45N, E45H, M46V, M46I, D48Q, D48N, L49V, L49I, E51Q, E51N, E51H, E60Q, E60N, E60H, K63Q, K63N, R67H, R67Q, P92S, P92A, E93Q, E93N, E93H, F119I, F119V, D120Q, D120N, E123Q, E123N, E123H, P124S and P124A; and
the one or more amino acid replacements occur in a mature GM-CSF cytokine having the sequence set forth in SEQ ID NO: 202 or in a sequence-related GM-CSF cytokine at a corresponding amino acid position(s) relative to SEQ ID NO: 202.
3 . The modified GM-CSF cytokine of claim 1 , wherein the unmodified GM-CSF cytokine contains the amino acid residues having the sequence set forth in SEQ ID NO: 202.
4 . The GM-CSF 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 GM-CSF cytokine of claim 2 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.
6 . The GM-CSF cytokine of claim 1 , wherein only the primary amino acid sequence is modified, and the GM-CSF cytokine exhibits increased resistance to proteolysis.
7 . The GM-CSF cytokine of claim 1 that exhibits increased protein half-life in vitro or in vivo compared to the unmodified GM-CSF cytokine.
8 . The GM-CSF cytokine of claim 1 that exhibits increased biological activity compared to the unmodified GM-CSF cytokine.
9 . The GM-CSF cytokine of claim 1 that exhibits comparable biological activity compared to the unmodified GM-CSF cytokine.
10 . The GM-CSF cytokine of claim 1 that exhibits decreased biological activity compared to the unmodified GM-CSF cytokine.
11 . A modified GM-CSF cytokine of claim 1 , comprising two or more amino acid replacements in its sequence of amino acid residues, wherein:
the modified GM-CSF cytokine exhibits increased resistance to proteolysis compared to the unmodified GM-CSF 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: E38Q, E38N, E38H, E41Q, E41N, E41H, E45Q, E45N, E45H, M46V, M46I, D48Q, D48N, L49V, L49I, E51Q, E51N, E51H, E60Q, E60N, E60H, K63Q, K63N, R67H, R67Q, P92S, P92A, E93Q, E93N, E93H, F119I, F119V, D120Q, D120N, E123Q, E123N, E123H, P124S and P124A; and
the one or more amino acid replacements occur in a mature GM-CSF cytokine having the sequence set forth in SEQ ID NO: 202 or in a sequence-related GM-CSF cytokine at corresponding amino acid position(s) relative to SEQ ID NO: 202.
12 . A nucleic acid molecule encoding a modified GM-CSF cytokine of claim 1 .
13 . A vector, comprising a nucleic acid molecule of claim 12 .
14 . A eukaryotic cell, comprising the nucleic acid molecule of claim 12 .
15 . A eukaryotic cell, comprising the vector of claim 13 .
16 . A collection of nucleic acid molecules, comprising a plurality of molecules of claim 12 .
17 . A collection of vectors, comprising a plurality of vectors of claim 13 .
18 . A method for expression of a modified GM-CSF cytokine, comprising:
introducing a nucleic acid of claim 12 into a host cell; and
culturing the cell under conditions, whereby the encoded modified GM-CSF cytokine is expressed.
19 . The method of claim 18 , further comprising isolating the modified GM-CSF cytokine.
20 . The method of claim 18 , wherein the host cell is a eukaryotic host cell or a bacterial cell.
21 . A pharmaceutical composition, comprising a GM-CSF cytokine of claim 1 .
22 . The pharmaceutical composition of claim 21 , further comprising a pharmaceutically acceptable carrier or excipient.
23 . The pharmaceutical composition of claim 22 , wherein the pharmaceutically acceptable carrier or excipient is selected from among a binding agent, a filler, a lubricant, a disintegrant, and a wetting agent.
24 . The pharmaceutical composition of claim 21 , further comprising a pharmaceutically acceptable additive.
25 . The pharmaceutical composition of claim 24 , wherein the pharmaceutically acceptable additive is selected from among a suspending agent, an emulsifying agent, a non-aqueous vehicle, and a preservative.
26 . The pharmaceutical composition of claim 21 , wherein the composition is in the form of a liquid, a solution, a suspension, an aerosol, a tablet, a lozenge or a capsule.
27 . The pharmaceutical composition of claim 26 , wherein the composition is in the form of a tablet or a capsule.
28 . The pharmaceutical composition of claim 21 , formulated for oral, parenteral, intravenous, intradermal, subcutaneous, buccal, inhalation, intramuscular, rectal or topical administration.
29 . The pharmaceutical composition of claim 28 , formulated for oral administration.
30 . The pharmaceutical composition of claim 29 , wherein the pharmaceutical composition is formulated for oral administration to the mouth or gastrointestinal tract.
31 . The pharmaceutical composition of claim 21 , wherein the pharmaceutical composition is formulated for controlled-release of the GM-CSF cytokine.
32 . A pharmaceutical composition formulated for oral administration, comprising a GM-CSF cytokine that contains one or more amino acid modification(s), whereby the GM-CSF cytokine exhibits increased protease resistance compared to a GM-CSF cytokine that does not contain the modification(s).
33 . The pharmaceutical composition of claim 32 , wherein the modified GM-CSF cytokine is 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.
34 . The pharmaceutical composition of claim 32 , wherein:
the modified GM-CSF cytokine comprises one or more amino acid replacements at one or more amino acid target positions in the unmodified cytokine.
35 . The pharmaceutical composition of claim 34 , wherein:
the modified GM-CSF cytokine comprises one or more amino acid replacements selected from among replacement of: E38Q, E38N, E38H, E41Q, E41N, E41H, E45Q, E45N, E45H, M46V, M46I, D48Q, D48N, L49V, L49I, E51Q, E51N, E51H, E60Q, E60N, E60H, K63Q, K63N, R67H, R67Q, P92S, P92A, E93Q, E93N, E93H, F119I, F119V, D120Q, D120N, E123Q, E123N, E123H, P124S and P124A; and
the one or more amino acid replacements occur in a mature GM-CSF cytokine having the sequence set forth in SEQ ID NO: 202 or in a sequence-related GM-CSF cytokine at corresponding amino acid position(s) relative to SEQ ID NO: 202.
36 . The pharmaceutical composition of claim 35 , wherein the GM-CSF cytokine comprises the sequence of amino acids set forth in any of SEQ ID NOS:362-400.
37 . The pharmaceutical composition of claim 35 , wherein the GM-CSF 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.
38 . The pharmaceutical composition of claim 32 , wherein the composition is in the form of a liquid, a solution, a suspension, an aerosol, a tablet, a lozenge or a capsule.
39 . The pharmaceutical composition of claim 38 , wherein the composition is in the form of a tablet or a capsule.
40 . The pharmaceutical composition of claim 32 , wherein the pharmaceutical composition is formulated for controlled-release of the GM-CSF cytokine.
41 . The pharmaceutical composition of claim 32 , wherein the GM-CSF cytokine exhibits increased resistance to proteolysis by a protease of the gastrointestinal tract.
42 . The pharmaceutical composition of claim 32 , wherein the GM-CSF cytokine exhibits increased biological activity compared to the unmodified GM-CSF cytokine.
43 . The pharmaceutical composition of claim 32 , wherein the GM-CSF cytokine exhibits comparable biological activity compared to the unmodified GM-CSF cytokine.
44 . The pharmaceutical composition of claim 32 , wherein the GM-CSF cytokine exhibits decreased biological activity compared to the unmodified GM-CSF cytokine.
45 . A pharmaceutical composition, comprising a nucleic acid molecule of claim 12 .
46 . A method, comprising treating a subject by administering the pharmaceutical composition of claim 21 , wherein the subject has a disease or condition that is treated by administration of a GM-CSF cytokine.
47 . The method of claim 46 , wherein the disease or condition is cancer or an autoimmune disease.
48 . The method of claim 47 , wherein the cancer is selected from among leukemia, melanoma, breast cancer, liver cancer and renal cancer.
49 . A method, comprising treating a subject by orally administering the pharmaceutical composition of claim 32 , wherein the subject has a disease or condition that is treated by administration of a GM-CSF cytokine.
50 . The method of claim 49 , wherein the disease or condition is cancer or an autoimmune disease.
51 . The method of claim 50 , wherein the cancer is selected from among leukemia, melanoma, breast cancer, liver cancer and renal cancer.