Unstructured recombinant polymers and uses thereof

• US 20080039341A1
• Filed: 03/06/2007
• Published: 02/14/2008
• Est. Priority Date: 09/27/2005
• Status: Active Grant

• Current Assignee: Amunix Pharmaceuticals, Inc. (Sanofi )
• Original Assignee: Amunix Operating Inc.
• Inventors: Scholle, Michael, Gordon, Nathaniel, Stemmer, Willem, Schellenberger, Volker, Wang, Chia-wei, Crameri, Andreas, Popkov, Mikhail
• Granted Patent: US 7,855,279 B2
• Time in Patent Office: Days
• US Class Current: 506/13
• 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 13/12 of the kidneys
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• 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 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 14/47 from mammals
• 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 14/56 IFN-alpha
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 2319/31 : fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/35 : containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C07K 7/08 : having 12 to 20 amino acids gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68
• CPC Class Codes: C12N 15/1044 : Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures

Abstract

The present invention provides unstructured recombinant polymers (URPs) and proteins containing one or more of the URPs. The present invention also provides microproteins, toxins and other related proteinaceous entities, as well as genetic packages displaying these entities. The present invention also provides recombinant polypeptides including vectors encoding the subject proteinaceous entities, as well as host cells comprising the vectors. The subject compositions have a variety of utilities including a range of pharmaceutical applications.

Claims

1. An unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein said URP is substantially incapable of non-specific binding to a serum protein, and wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

3. The URP of claim 1 or 2, wherein the URP comprises a non-natural amino acid sequence.

4. The URP of claim 1 or 2, wherein the URP is selected for incorporation into a heterologous protein, and wherein upon incorporation the URP into a heterologous protein, said heterologous protein exhibits a longer serum half-life and/or higher solubility as compared to the corresponding protein that is deficient in said URP.

5. The URP of claim 1 or 2, wherein upon incorporation of the URP into a heterologous protein, said heterologous protein exhibits a serum secretion half-life that is at least two times longer as compared to the corresponding protein that is deficient in said URP.

6. The URP of claim 1 or 2, wherein incorporation of the URP into a heterologous protein results in at least a 2-fold increase in apparent molecular weight of the protein as approximated by size exclusion chromatography.

7. The URP of claim 1 or 2, wherein the URP has a Tepitope score less than −

4.

8. The URP of claim 1 or 2, wherein the amino acids are predominantly hydrophilic residues.

9. The URP of claim 1 or 2, wherein at least 50% of the amino acids of the URP are devoid of secondary structure as determined by Chou-Fasman algorithm.

10. The URP of claim 1 or 2, wherein glycine residues contained in the URP constitute at least about 50% of the total amino acids of the URP.

11. The URP of claim 1 or 2, wherein any one type of the amino acids alone selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) constitutes more than about 20% of the total amino acids of the URP.

12. The URP of claim 1 or 2, wherein any one type of the amino acids alone selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) constitutes more than about 40% of the total amino acids of the URP.

13. The URP of claim 1 or 2, wherein the URP comprises more than about 100 contiguous amino acids.

14. The URP of claim 1 or 2, wherein the URP comprises more than about 200 contiguous amino acids.

15. The URP of claim 1 or 2 comprising repeat sequences.

16. The URP of claim 1 or 2, wherein one type of the amino acids alone selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P), constitutes more than 50% of the total amino acids of the URP.

17. A protein comprising one or more URPs of claim 1 or 2, wherein said one or more URPs are heterologous with respect to the protein.

18. The protein of claim 17 comprising an effector module.

19. The protein of claim 17 comprising a binding module.

20. The protein of claim 17 comprising an effector module and a binding module.

21. The protein of claim 17 comprising a plurality of binding modules, wherein the individual binding modules exhibit binding specificities to the same or different targets.

22. The protein of claim 17, wherein the total length of URPs in aggregation exceeds about 150 amino acids.

23. The protein of claim 17 comprising one or more binding modules, wherein the binding module comprises a disulfide-containing scaffold formed by intra-scaffold pairing of cysteines.

24. The protein of claim 17 comprising an effector module which is cytotoxic.

25. The protein of claim 17 comprising a binding module specific for a target molecule, wherein the target is selected from the group consisting of cell surface protein, secreted protein, cytosolic protein, and nuclear protein.

26. The protein of claim 17 comprising a binding module specific for a target molecule, wherein the target is an ion channel.

27. The protein of claim 17 exhibiting an extended serum secretion half-life by at least 2 folds as compared to a corresponding protein that is deficient in said URP.

39. A recombinant polynucleotide comprising a coding sequence that encodes the URP of claim 1 or 2.

40. A recombinant polynucleotide comprising a coding sequence that encodes the protein of claim 17.

42. A host cell comprising the recombinant polynucleotides of claim 40.

44. A vector comprising the recombinant polynucleotide of claim 40.

46. A selectable library of expression vectors comprising more than one vector of claim 44.

48. A genetic package displaying the library of claim 46.

2. An unstructured recombinant polymer (URP) comprising at least 40 contiguous amino acids, wherein said URP has an in vitro serum degradation half-life greater than about 24 hours, and wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

28. A non-naturally occurring protein comprising at least 3 repeating units of amino acid sequences, each of the repeating unit comprising at least 6 amino acids, wherein the majority of segments comprising about 6 to about 15 contiguous amino acids of the at least 3 repeating units are present in one or more native human proteins.

29. The protein of claim 28, wherein the majority of segments comprising about 9 to about 15 contiguous amino acids within the repeating units are present in one or more native human proteins.

30. The protein of claim 28, wherein each segment comprising about 6 to about 15 contiguous amino acids within the protein, is present in at least one native human protein.

31. The protein of claim 28, wherein each segment comprising about 9 to about 15 contiguous amino acids within the protein, is present in at least one native human protein.

32. The protein of claim 28, wherein the at least 3 repeating units share sequence identity of greater than about 80%.

33. The protein of claim 28, wherein each of the repeating units comprises about 6 to about 15 contiguous amino acids.

34. The protein of claim 28, wherein each of the repeating units comprises about 9 contiguous amino acids.

35. The protein of claim 28 comprising one or more modules selected from the group consisting of binding modules, effector modules, multimerization modules, C-terminal modules, and N-terminal modules.

36. The protein of claim 28, wherein an individual repeating unit comprises an unstructured recombinant polymer (URP).

37. The protein of claim 36, wherein the URP comprises at least 40 contiguous amino acids, wherein said URP is substantially incapable of non-specific binding to a serum protein, and wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

38. The protein of claim 36, wherein the URP comprises at least 40 contiguous amino acids, wherein said URP has an in vitro serum degradation half-life greater than about 24 hours, and wherein

(a) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes more than about 80% of the total amino acids of the URP;

and/or

(b) at least 50% of the amino acids are devoid of secondary structure as determined by Chou-Fasman algorithm.

41. A recombinant polynucleotide comprising a coding sequence that encodes the protein of claim 28.

43. A host cell comprising the recombinant polynucleotides of claim 41.

45. A vector comprising the recombinant polynucleotide of claim 41.

47. A selectable library of expression vectors comprising more than one vector of claim 45.

49. A genetic package displaying the library of claim 47.