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

• US 20070224665A1
• Filed: 02/14/2007
• Published: 09/27/2007
• Est. Priority Date: 09/09/2002
• Status: Active Grant

• Current Assignee: HanAll Biopharma Co. Ltd.
• Original Assignee: HanAll Biopharma Co. Ltd.
• Inventors: Guyon, Thierry, Drittanti, Lila, Vega, Manuel, Gantier, Rene
• Granted Patent: US 7,650,243 B2
• Time in Patent Office: Days
• US Class Current: 435/69.510
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/53 Colony-stimulating factor [CSF]
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/54 Interleukins [IL]
• CPC Class Codes: C07K 14/555 : Interferons [IFN]
• CPC Class Codes: C07K 14/575 : Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695
• CPC Class Codes: C07K 14/61 : Growth hormone [GH], i.e. somatotropin

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.

Claims

1. A method for generating a protein or peptide molecule, having a predetermined property or activity, the method comprising:

(a) identifying, within a target protein or peptide or plurality thereof, one or more target amino acids, wherein;

each target amino acid is designated an in silico-HIT (is-HIT); and

the is-HIT target amino acids are identified by identifying structurally homologous loci between the target protein and a reference protein possessing the desired activity;

(b) identifying one or more replacement amino acids, specific for each is-HIT, wherein each protein or peptide comprising a single amino acid replacement within the target protein or peptide is designated as a candidate LEAD protein;

(c) producing a population of separate sets of nucleic acid molecules, wherein;

each set encodes a different candidate LEAD protein;

all nucleic acid molecules in a set encode the same protein;

each candidate LEAD protein contains a single amino acid replacement; and

each set of nucleic acid molecules encodes a candidate LEAD protein that differs by one amino acid from the target protein or peptide;

(d) separately introducing each set of nucleic acid molecules into host cells and expressing the encoded candidate LEAD proteins, wherein the host cells are addressably arrayed; and

(e) individually screening each set of encoded candidate LEAD proteins to identify one or more proteins that has an activity that differs from an activity an unmodified target protein, wherein each such protein is designated a LEAD mutant protein.

2. The method of claim 1, wherein the predetermined property or activity is selected from among resistance to proteolysis, resistance to glomerular filtration, conformational stability, catalytic activity, and immunogenicity.

3. The method of claim 1, wherein the predetermined property or activity is increased resistance to proteolysis.

4. The method of claim 1, wherein the target protein is a cytokine.

5. The method of claim 4, wherein the cytokine is selected from among interleukin-10 (IL-10), interferon beta (IFNβ

), interferon alpha (IFNα

), 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 method of claim 1, wherein each candidate LEAD protein is individually prepared and screened to identify LEAD mutant proteins.

7. The method of claim 1, wherein the nucleic acid molecules comprise plasmids.

8. The method of claim 1, wherein the host cells are eukaryotic cells or bacterial cells.

9. The method of claim 1, wherein the nucleic acid molecules in step (c) are produced by site-specific mutagenesis.

10. The method of claim 1, further comprising:

(f) generating a population of sets of nucleic acid molecules encoding a set of candidate super-LEAD proteins, wherein;

each candidate super-LEAD protein comprises a combination of two or more of the single amino acid replacements derived from two or more LEAD mutant proteins;

each set encodes a different candidate super-LEAD protein; and

all nucleic acid molecules in a set encode the same protein;

(g) introducing each set of nucleic acid molecules encoding candidate super-LEAD proteins into cells and expressing the encoded candidate super-LEAD proteins; and

(h) individually screening each set of encoded candidate super-LEAD proteins to identify one or more proteins that has activity that differs from the unmodified target protein and has properties that differ from the original LEAD mutant proteins, wherein each such protein is designated a super-LEAD.

11. The method of claim 10 wherein the nucleic acid molecules in step (f) are produced by a method selected from among recombination, mutagenesis, nucleic acid shuffling, additive directional mutagenesis (ADM), multi-overlapped primer extensions, oligonucleotide-mediated mutagenesis, and de novo synthesis.

12. The method of claim 1, wherein the replacement amino acids identified in step (b) correspond to a restricted subset of the 19 remaining non-native amino acids.

13. The method of claim 1, wherein the nucleic acids of step (c) are produced by systematically replacing each codon that is an is-HIT, with a codon encoding an amino acid selected from among a restricted subset of the 19 remaining non-native amino acids, to produce nucleic acid molecules each differing by at least one codon and encoding candidate LEAD proteins.

14. The method of claim 10, wherein the number of LEAD amino acid positions generated on a single nucleic acid molecule is selected from the group consisting of:

two, three, four, five, six, seven, eight, nine, ten or more LEAD amino acid positions up to all of the LEAD amino acid positions.

15. The method of claim 1, wherein the LEAD mutant protein possesses increased resistance to proteolysis compared to the unmodified target protein.

16. The method of claim 10, wherein the super-LEAD possesses increased resistance to proteolysis compared to the unmodified target protein.

17. The method of claim 1, wherein the LEAD mutant protein possesses increased conformational stability compared to the unmodified target protein.

18. The method of claim 10, wherein the super-LEAD possesses increased conformational stability compared to the unmodified target protein.

19. The method of claim 1, wherein the replacement amino acids are selected using Percent Accepted Mutations (PAM) matrices.

20. The method of claim 1, wherein identification of the structurally homologous loci between the target protein and a reference protein possessing the desired activity, comprises:

(a) comparing the 3-dimensional structures of the two or more proteins to identify regions of high coincidence between their backbones, said regions designated as structurally homologous regions; and

(b) identifying is-HIT structurally homologous loci on the evolving target protein that correspond to structurally related is-HIT amino acid positions within a structurally homologous region of the reference protein.

21. The method of claim 20, wherein the evolving target protein and the reference protein are sequence-related proteins.

22. The method of claim 20, wherein the evolving target protein and the reference protein are non-related proteins or sequence-non-related proteins.

23. The method of claim 20, wherein the degree of coincidence between the 3-dimensional structures of the evolving target protein and the reference protein is in a region selected from the group consisting of:

(a) a region that is less than about 10%, 15%, 25% of one or both of the two proteins;

(b) a region that is greater than about 10%, 15% or 25% of one or both of the two proteins; and

(c) a region that covers the full length of one or both of the two proteins.

24. The method of claim 20, wherein the degree of coincidence between the 3-dimensional structures of the evolving target protein and of the reference protein is determined by superposition and root means squared (RMS) deviation calculations using any combination of one or more of the peptide backbone atoms selected from the group consisting of:

N, C, C(C═

O), O and CA.

25. The method of claim 24, wherein the superposition and RMS deviation calculations are made using all of the peptide backbone atoms selected from the group consisting of:

N, C, C(C═

O), O and CA.

26. The method of claim 24, wherein the superposition and RMS deviation calculations are carried out on a subset of regions or domains of a larger protein that adopts a structure similar to a smaller protein.

27. The method of claim 20, wherein the degree of coincidence between the 3-dimensional structures of the evolving target protein and the reference protein is obtained using a method or a combination of two or more methods selected from among:

Class Architecture, Topology and Homologous Superfamily (CATH);

Combinatorial Extension of the optimal path (CE);

Fold Classification based of Structure-Structure Alignment of Proteins (FSSP);

Structural Classification of Proteins (SCOP);

Vector Alignment Search Tool (VAST), and TOP.

28. A method of generating a modified protein or cytokine having a predetermined property or activity, the method comprising:

(a) modifying a first protein or cytokine by a directed evolution method to produce an evolved protein or cytokine that has the predetermined property or activity to identify altered loci; and

(b) comparing the structures of one or more members of the protein or cytokine family to identify structurally homologous loci for alteration;

(c) altering the identified loci in members of the protein or cytokine family to produce proteins or cytokines that have the altered phenotype.

29. The method of claim 28, wherein the directed evolution method is a rational directed evolution method.

30. The method of claim 28, wherein the directed evolution method is a 2-dimensional rational scanning method.

31. The method of claim 28, wherein identification of the structurally homologous loci between the evolved protein or cytokine and members of the protein or cytokine family, comprises:

(a) comparing the 3-dimensional structures of the evolved protein or cytokine with one or more members of the protein or cytokine family to identify regions of high coincidence between their backbones, said regions designated as structurally homologous regions; and

(b) identifying is-HIT structurally homologous loci on the members of the protein or cytokine family that correspond to structurally related is-HIT amino acid positions within a structurally homologous region of the evolved protein or cytokine.

32. The method of claim 31, wherein the degree of coincidence between the 3-dimensional structures of the members of the protein or cytokine family and the evolved protein or cytokine is in a region selected from the group consisting of:

(a) a region that is less than about 10%, 15%, 25% of one or both of the two proteins;

(b) a region that is greater than about 10%, 15% or 25% of one or both of the two proteins; and

(c) a region that covers the full length of one or both of the two proteins.

33. The method of claim 31, wherein the degree of coincidence between the 3-dimensional structures of the members of the protein or cytokine family and of the evolved protein or cytokine is determined by superposition and RMS deviation calculations using any combination of one or more of the peptide backbone atoms selected from the group consisting of:

N, C, C(C═

O), O and CA.

34. The method of claim 33, wherein the superposition and RMS deviation calculations are made using all of the peptide backbone atoms present selected from group the consisting of:

N, C, C(C═

O), O and CA.

35. The method of claim 31, wherein the superposition and RMS deviation calculations are carried out on a subset of regions or domains of a larger protein that adopts a structure similar to a smaller protein.

36. The method of claim 31, wherein the degree of coincidence between the 3-dimensional structures of the members of the protein or cytokine family and the evolved protein or cytokine is obtained using a method or a combination of two or more methods selected from among:

Class Architecture, Topology and Homologous Superfamily (CATH);

Combinatorial Extension of the optimal path (CE);

Fold Classification based of Structure-Structure Alignment of Proteins (FSSP);

Structural Classification of Proteins (SCOP);

Vector Alignment Search Tool (VAST), and TOP.

37. The method of claim 28, wherein the 2-dimensional rational scanning method comprises:

(a) identifying, within the first protein or cytokine, one or more target amino acids amenable to providing the altered phenotype upon amino acid replacement, wherein each target amino acid is designated an in silico-HIT (is-HIT);

(b) identifying one or more replacement amino acids, specific for each is-HIT, amenable to providing the altered phenotype upon amino acid replacement, wherein each single amino acid replacement within the protein or cytokine is designated as a candidate LEAD protein;

(c) producing a population of sets of nucleic acid molecules that encode each of the candidate LEAD proteins, wherein each candidate LEAD protein comprises a single amino acid replacement, and wherein each polynucleotide in a set encodes a candidate LEAD protein that differs by one amino acid from the unmodified protein or cytokine;

(d) introducing each set of nucleic acid molecules into host cells and expressing the encoded candidate LEAD proteins, wherein the host cells are present in an addressable array; and

(e) individually screening the sets of encoded candidate LEAD proteins to identify one or more candidate LEAD proteins that has activity that differs from the unmodified protein or cytokine, wherein each such protein is designated a LEAD mutant protein.

38. The method of claim 37, wherein the array comprises a solid support with wells and each well contains one set of cells.

39. The method of claim 37, wherein the nucleic acid molecules comprise plasmids.

40. The method of claim 37, wherein the host cells are eukaryotic cells or bacterial cells.

41. The method of claim 37, wherein the nucleic acid molecules in step (c) are produced by site-specific mutagenesis.

42. The method of claim 37, wherein the LEAD mutant protein possesses increased resistance to proteolysis compared to the unmodified protein or cytokine.

43. The method of claim 37, further comprising:

(f) generating a population of sets of nucleic acid molecules encoding a set of candidate super-LEAD proteins, wherein each candidate super-LEAD protein comprises a combination of two or more of the single amino acid mutations derived from two or more LEAD mutant proteins;

(g) introducing each set of nucleic acid molecules encoding candidate super-LEADs into cells and expressing the encoded candidate super-LEAD proteins; and

(h) individually screening the sets of encoded candidate super-LEAD proteins to identify one or more proteins that has activity that differs from the unmodified protein or cytokine and has properties that differ from the original LEAD mutant proteins, wherein each such protein is designated a super-LEAD.

44. The method of claim 43 wherein the nucleic acid molecules in step (f) are produced by a method selected from among recombination, mutagenesis, nucleic acid shuffling, additive directional mutagenesis (ADM), multi-overlapped primer extensions, oligonucleotide-mediated mutagenesis, and de novo synthesis.

45. The method of claim 37, wherein the replacement amino acids identified in step (b) correspond to a restricted subset of the 19 remaining non-native amino acids.

46. The method of claim 37, wherein the nucleic acids of step (c) are produced by systematically replacing each codon that is an is-HIT, with a codon encoding an amino acid of a restricted subset of the 19 remaining non-native amino acids, to produce nucleic acid molecules each differing by at least one codon and encoding candidate LEAD proteins.

47. The method of claim 43, wherein the number of LEAD amino acid positions generated on a single nucleic acid molecule is selected from the group consisting of:

two, three, four, five, six, seven, eight, nine, ten or more LEAD amino acid positions up to all of the LEAD amino acid positions.