Proteinaceous pharmaceuticals and uses thereof

• US 20070212703A1
• Filed: 09/27/2006
• Published: 09/13/2007
• Est. Priority Date: 09/27/2005
• Status: Abandoned Application

• Current Assignee: Amunix Operating Inc.
• Original Assignee: Amunix Operating Inc.
• Inventors: Stemmer, Willem, Schellenberger, Volker, Bader, Martin, Scholle, Michael
• Application Number:
  US11/528,950
• Publication Number:
  US 20070212703A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 taraget 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 scaffold exhibiting a binding specificity towards a target molecule, comprising a polypeptide having two disulfide bonds formed by pairing intra-scaffold cysteines according to a pattern selected from the group consisting of C^{1-2 3-4}, C^{1-3, 2-4}, and C^{1-4, 2-3}, wherein the two numerical numbers linked by a hyphen indicate which two cysteines counting from N-terminus of the polypeptide are paired to form a disulfide bond.

4. The non-naturally occurring cysteine (C)-containing scaffold of claim 1, 2 or 3 that remains the target binding capability after being heated to a temperature higher than about 50°

C.

5. The non-naturally occurring cysteine (C)-containing scaffold of claim 1, 2 or 3 that remains the target binding capability after being heated to a temperature higher than about 80°

C.

6. The non-naturally occurring cysteine (C)-containing scaffold of claim 1, 2 or 3 that remains the target binding capability after being heated to a temperature higher than about 100°

C. and for more than 0.1 second.

7. The non-naturally occurring cysteine (C)-containing scaffold of claim 1, 2 or 3 that is conjugated to a moiety selected from the group consisting of labels, effectors, and antibodies.

8. The non-naturally occurring cysteine (C)-containing scaffold of claim 1, 2 or 3 being a monomer.

9. The non-naturally occurring cysteine (C)-containing scaffold of claim 1, 2 or 3 comprising a half-life extrension moiety.

10. The non-naturally occurring cysteine (C)-containing scaffold of claim 9, wherein the half-life extrension moiety selected from the group consisting of serum albumin, IgG, erythrocytes, and and proteins accessible to the serum.

11. The non-naturally occurring cysteine (C)-containing scaffold of claim 1, 2 or 3 exhibiting binding specificity towards a target distinct from the native target of the corresponding nacturally-occurring cysteine (C)-containing protein or scaffold.

12. A library of the non-naturally occurring cysteine (C)-containing scaffold of claim 1, 2 or 3.

13. A genetic package displaying the library of claim 12.

14. 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 13;

(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.

15. The method of claim 14 further comprising the step of isolating the genetic package that displays a polypeptide having the desired property.

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

17. The method of claim 12, wherein the page is filamentous phage.

18. A method of producing a non-naturally occurring cysteine (C)-containing scaffold, comprising:

providing a host cell comprising a nucleic acid encoding a a non-naturally occurring cysteine (C)-containing scaffold of any one of claims 1-3;

culturing said host cell in a suitable culture medium under conditions to effect expression of said scaffold from said nucleic acid.

19. The method of claim 14 further comprising the step of recovering said scaffold from said medium.

20. A pharmaceutical composition comprising the non-naturally occurring cysteine (C)-containing scaffold of claim 1, 2 or 3 and a pharmaceutically acceptable carrier.

2. A non-naturally occurring cysteine (C)-containing scaffold exhibiting a binding specificity towards a target molecule, comprising a polypeptide having three disulfide bonds formed by pairing intra-scaffold cysteines according to a pattern selected from the group consisting of C^{1-2, 3-4, 5-6}, C^{1-2, 3-5, 4-6}, C^{1-2, 3-6, 4-5}, C^{1-2, 3-6, 5-6}, C1^{-3, 2-5, 4-6}, C^{1-3, 2-6, 4-5}, C^{1-4, 2-3, 5-6}, C^{1-4, 2-6, 3-5}, C^{1-5, 2-3, 4-6}, C^{1-5, 2-4, 3-6}, C^{1-5, 2-6, 3-4}, C^{1-6, 2-3, 4-5}, and C^{1-6, 2-5, 3-4}, wherein the two numerical numbers linked by a hyphen indicate which two cysteines counting from N-terminus of the polypeptide are paired to form a disulfide bond.

3. A non-naturally occurring cysteine (C)-containing scaffold exhibiting a binding specificity towards a target molecule, comprising a polypeptide having at least four disulfide bonds formed by pairing intra-scaffold cysteines according to a pattern selected from the following: