Unstructured recombinant polymers and compositions comprising same

• US 8,492,530 B2
• Filed: 11/03/2010
• Issued: 07/23/2013
• Est. Priority Date: 09/27/2005
• Status: Active Grant

• Current Assignee: Amunix Pharmaceuticals, Inc. (Sanofi )
• Original Assignee: Amunix Operating Inc.
• Inventors: Schellenberger, Volker, Stemmer, Willem P., Wang, Chia-Wei, Scholle, Michael D., Popkov, Mikhail, Gordon, Nathaniel C., Crameri, Andreas
• Primary Examiner(s): WESSENDORF, TERESA D
• Application Number:
  US12/939,120
• Publication Number:
  US 20110171687A1
• Time in Patent Office: 993 Days
• Field of Search: 530/399, 530/350, 530/324, 530/300, 530/417, 435/4
• US Class Current: 530/399
• 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. A fusion protein comprising at least one unstructured recombinant polypeptide (URP) and a heterologous protein, wherein the heterologous protein is a pharmaceutically active protein selected from the group consisting of cytokines, growth factors, enzymes, receptors, microproteins, hormones, toxins, and hirudin, wherein the fusion protein exhibits an increased serum half-life as compared to the heterologous protein lacking said URP, and wherein the at least one URP is characterized in that:

(a) it comprises at least 40 contiguous amino acids;

(b) 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 80% of the total amino acids of the URP, and the remainder, when present, consists of Arginine or Lysine, and the remainder does not contain methionine, cysteine, asparagine, and glutamine;

(c) the URP comprises at least three different types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), wherein one of said at least three different types is glycine (G) or glutamine (E);

(d) at least 50% of the at least 40 contiguous amino acids in said at least one URP sequence are devoid of secondary structure as determined by Chou-Fasman algorithm; and

(e) the URP has a Tepitope score less than −

5.

2. The fusion protein of claim 1, wherein the at least one URP is fused into the heterologous protein at an N-terminus, a C-terminus, into a loop of the heterologous protein, or between domains of the heterologous protein, or into both the N-terminus and the C-terminus of the heterologous protein.

3. The fusion protein of claim 1, wherein the at least one URP is fused into a loop of the heterologous protein or between domains of the heterologous protein.

4. The fusion protein of claim 1, exhibiting at least a 2-fold increase in apparent molecular weight as compared to the heterologous protein lacking the at least one URP, wherein the apparent molecular weight is approximated by size exclusion chromatography.

5. The fusion protein of claim 1, wherein the fusion protein exhibits a serum secretion half-life that is at least four-fold longer as compared to the heterologous protein lacking said at least one URP.

6. The fusion protein of claim 1, wherein one of the amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) alone constitutes more than 20% of the total amino acids said at least one URP.

7. The fusion protein of claim 1, wherein one of the amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) alone constitutes more than 30% of the total amino acids of said at least one URP.

8. The fusion protein of claim 1, wherein any one type of amino acid is no more than 30% of the total amino acids of the URP.

9. The fusion protein of claim 1, wherein the at least one URP comprises at least five types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).

10. The fusion protein of claim 1, wherein the at least one URP comprises at least six types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).

11. The fusion protein of claim 1, wherein the heterologous protein is selected from the group consisting of erythopoetin, interferon, human growth hormone, growth hormone releasing hormone, G-CSF, GM-CSM, insulin, hirudin, TNF-receptor, uricase, thrombin, blood clotting enzyme, VEGF, protropin, somatropin, alteplase, interleukin, factor VII, factor VIII, factor X, factor IX, dornase, glucocerebrosidase, follitropin, and glucagon.

12. The fusion protein of claim 11, wherein the heterologous protein is a human growth hormone.

13. The fusion protein of claim 11, wherein the heterologous protein is a blood clotting enzyme.

14. The fusion protein of claim 11, wherein the heterologous protein is factor VII.

15. The fusion protein of claim 11, wherein the heterologous protein is factor VIII.

16. The fusion protein of claim 11, wherein the heterologous protein is factor IX.

17. The fusion protein of claim 11, wherein the heterologous protein is insulin.

18. The fusion protein of claim 1, wherein the combined length of the at least one URP exceeds 100 amino acids.

19. The fusion protein of claim 1, wherein the combined length of the at least one URP exceeds 200 amino acids.

20. The fusion protein of claim 1, wherein the at least one URP comprises more than 100 contiguous amino acids.

21. The fusion protein of claim 1, wherein the at least one URP comprises more than 200 contiguous amino acids.

22. A method of producing the fusion protein, comprising:

(a) providing a host cell comprising an expression vector comprising a polynucleotide encoding the fusion protein of claim 1;

(b) culturing said host cell in a culture medium under conditions effective to effect expression of said fusion protein from said polynucleotide;

(c) adsorbing said fusion protein onto a chromatography substrate under conditions effective to capture said fusion protein onto the chromatography substrate;

(d) eluting said fusion protein under conditions effective to elute said capture fusion protein; and

(e) recovering said fusion protein.

23. The method of claim 22, wherein the heterologous protein is selected from the group consisting of erythopoetin, interferon, human growth hormone, growth hormone releasing hormone, G-CSF, GM-CSM, insulin, hirudin, TNF-receptor, uricase, thrombin, blood clotting enzyme, VEGF, protropin, somatropin, alteplase, interleukin, factor VII, factor VIII, factor X, factor IX, dornase, glucocerebrosidase, follitropin, and glucagon.

24. The method of claim 22, wherein the host cell is a eukaryotic cell.

25. The method of claim 22, wherein the host cell is a prokaryotic cell.

26. The fusion protein of claim 1, wherein the URP comprises at least four types of amino acids selected from the group consisting of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P).

27. The fusion protein of claim 1, wherein glutamate constitutes at least 5% of the total amino acids of the URP.