Methods for production of unstructured recombinant polymers and uses thereof

• US 20080286808A1
• Filed: 03/06/2007
• Published: 11/20/2008
• Est. Priority Date: 09/27/2005
• Status: Active Grant

• Current Assignee: Amunix Pharmaceuticals, Inc. (Sanofi )
• Original Assignee: Amunix Operating Inc.
• Inventors: Schellenberger, Volker, Wang, Chia-wei, Stemmer, Willem P., Crameri, Andreas, Scholle, Michael D., Popkov, Mikhail, Gordon, Nathaniel C.
• Granted Patent: US 7,846,445 B2
• Time in Patent Office: Days
• US Class Current: 435/7.8
• 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: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/43 containing a FLAG-tag
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C12N 2795/14011 ssDNA Bacteriophages
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• 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 method of producing a protein comprising an unstructured recombinant polymer (URP), comprising:

(i) providing a host cell comprising a recombinant polynucleotide encoding the protein, said protein comprising one or more URP, said 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;

(ii) culturing said host cell in a suitable culture medium under conditions to effect expression of said protein from said polynucleotide.

2. The method of claim 1 wherein the URP has an in vitro serum degradation half-life greater than about 24 hours.

3. The method of claim 1 wherein the host cell is a eukaryotic cell.

4. The method of claim 1 wherein the host cell is CHO cell.

5. The method of claim 1 wherein the host cell is a prokaryotic cell.

16. The method of claim 1, 6 or 8, wherein the URP comprises a non-natural amino acid sequence.

17. The method of claim 1, 6 or 8, wherein the URP has a Tepitope score equal to or less than −

4.

18. The method of claim 1, 6 or 8, wherein the URP is devoid of secondary structure as determined by Chou-Fasman algorithm.

19. The method of claim 1, 6 or 8, wherein glycine residues contained in the URP constitute at least about 50% of the total amino acids of the URP.

20. The method of claim 1, 6 or 8, wherein 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 60% of the total amino acids of the URP.

21. The method of claim 1, 6, or 8, wherein one type 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) constitutes more than 50% of the total amino acids of the URP.

22. The method of claim 1, 6 or 8, wherein the URP comprises more than 100 contiguous amino acids.

23. The method of claim 1, 6 or 8, wherein the URP comprises repeat sequences.

24. The method of claim 1, 6 or 8, wherein the protein is a therapeutic protein.

25. The method of claim 1, 6 or 8, wherein the protein comprises one or more modules selected from the group consisting of binding modules, effector modules, multimerization modules, C-terminal modules, and N-terminal modules.

6. A method of increasing serum secretion half-life of a protein, comprising:

fusing said protein with one or more unstructured recombinant polymers (URPs), wherein the URP comprises at least about 40 contiguous amino acids, 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; and

wherein said URP is substantially incapable of non-specific binding to a serum protein.

7. The method of claim 6 wherein the serum secretion half-life of the protein is extended by at least 2 folds.

8. A method of detecting the presence or absence of a specific interaction between a target and an exogenous protein that is displayed on a genetic package, wherein said protein comprises one or more unstructured recombinant polymer (URP), the method comprising:

(a) providing a genetic package displaying a protein that comprises one or more unstructured recombinant polymers (URPs);

(b) contacting the genetic package with the target under conditions suitable to produce a stable protein-target complex; and

(c) detecting the formation of the stable protein-target complex on the genetic package, thereby detecting the presence of a specific interaction.

9. The method of claim 8 further comprises obtaining a nucleotide sequence from the genetic package that encodes the exogenous protein.

10. The method of claim 8, wherein the presence or absence of a specific interaction is between the URP and a target comprising a serum protein.

11. The method of claim 8, wherein the presence or absence of a specific interaction is between the URP and a target comprising a serum protease.

12. The method of claim 8, wherein the target or the protein is selected from the group consisting of cell surface protein, secreted protein, cytosolic protein, and nuclear protein:

13. The method of claim 8, wherein the genetic package is phage.

14. The method of claim 8, wherein the URP comprises at least about 40 contiguous amino acids, and wherein the URP is substantially incapable of non-specific binding to a serum protein, and further 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;

and/or

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

15. The method of claim 8, wherein the URP comprises at least about 40 contiguous amino acids, and wherein the URP has an in vitro serum degradation half-life greater than about 24 hours.