Proteinaceous pharmaceuticals and uses thereof

• US 20070191272A1
• Filed: 09/27/2006
• Published: 08/16/2007
• Est. Priority Date: 09/27/2005
• Status: Abandoned Application

• Current Assignee: Amunix Operating Inc.
• Original Assignee: Amunix Operating Inc.
• Inventors: Stemmer, Willem, Schellenberger, Volker, Scholle, Michael, Bader, Martin
• Application Number:
  US11/528,927
• Publication Number:
  US 20070191272A1
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: A61K 38/16 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof enzyme inhibitors A61K38/005
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 14/415 from plants
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• 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 cysteine-containing scaffolds and/or proteins, expression vectors, host cell and display systems harboring and/or expressing such cysteine-containing products. The present invention also provides methods of designing libraries of such products, methods of screening such libraries to yield entities exhibiting binding specificities towards a target molecule. Further provided by the invention are pharmaceutical compositions comprising the cysteine-containing products of the present invention.

Claims

1. A non-naturally occurring cysteine (C)-containing protein comprising a polypeptide having no more than 35 amino acids, in which

at least 10% of the amino acids in the polypeptide are cysteines,

at least two disulfide bonds are formed by pairing intra-scaffold cysteines, and wherein said pairing yields a complexity index greater than 3.

3. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2, wherein the complexity index greater than 6.

4. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2, wherein the complexity index greater than 10.

5. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2 that binds specifically to a target molecule.

6. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2 that remains the target binding capability after being heated to a temperature higher than about 50°

C.

7. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2 that remains the target binding capability after being heated to a temperature higher than about 80°

C.

8. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2 that remains the target binding capability after being heated to a temperature higher than about 100°

C. and for more than 0.1 second.

9. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2 that is conjugated to a moiety selected from the group consisting of labels, effectors, antibodies, and half-life extending moieties.

10. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2 being a monomer.

11. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2 being a multimer.

12. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2, wherein the protein comprises one type of scaffold.

13. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2, wherein the protein comprises more than one type of scaffold.

14. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2, wherein the protein comprises a target binding site and half-life extension moiety.

15. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2, wherein the protein comprises repeating units that bind to the target.

16. The non-naturally occurring cysteine (C)-containing protein of claim 1 or 2, wherein the protein comprises a half-life extension moiety selected from the group consisting of serum albumin, IgG, erythrocytes, and proteins accessible to the serum.

20. A library of the non-naturally occurring protein of claim 1, 2, 18 or 19.

21. A genetic package displaying the library of claim 20.

22. A method of detecting the presence of a specific interaction between a target and an exogenous polypeptide that is displayed on a genetic package, the method comprising:

(a) providing a genetic package displaying of claim 20;

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

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

23. The method of claim 22 further comprising the step of isolating the genetic package that displays a polypeptide having the desired property.

24. A pharmaceutical composition comprising the non-naturally occurring cysteine (C)-containing protein of claim 1 or 2 and a pharmaceutically acceptable carrier.

2. A non-naturally occurring cysteine (C)-containing protein comprising a polypeptide having no more than about 60 amino acids, in which

at least 10% of the amino acids in the polypeptide are cysteines,

at least four disulfide bonds are formed by pairing cysteines contained in the polypeptide, and wherein said pairing yields a complexity index greater than 4.

17. The non-naturally occurring cysteine (C)-containing protein exhibiting binding specificity towards a target distinct from the native target of the corresponding naturally-occurring cysteine (C)-containing protein or scaffold.

18. A non-natural protein containing a single domain of 20-60 amino acids which has 3 or more disulfides and binds to a human serum-exposed protein, and wherein said protein has less than 5% aliphatic amino acids.

19. A non-naturally occurring protein containing a single domain of 20-60 amino acids which has 3 or more disulfides and binds to a human serum-exposed protein, wherein said protein has a score in the T-Epitope program that is less than 90% of the average for proteins in the database.