Protein scaffolds

• US 8,633,297 B2
• Filed: 10/31/2008
• Issued: 01/21/2014
• Est. Priority Date: 10/31/2007
• Status: Active Grant

• Current Assignee: Medimmune LLC (Astrazeneca PLC)
• Original Assignee: Medimmune LLC (Astrazeneca PLC)
• Inventors: Wu, Herren, Baca, Manuel, Swers, Jeffrey, Chacko, Benoy
• Primary Examiner(s): Steele, Amber D
• Application Number:
  US12/739,149
• Publication Number:
  US 20100298541A1
• Time in Patent Office: 1,908 Days
• Field of Search: 530/300, 514/1.1
• US Class Current: 530/300
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 17/14 for baldness or alopecia
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 27/06 Antiglaucoma agents or miotics
• 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 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 7/06 : Antianaemics
• 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 14/78 : Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 16/2878 : against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 2318/20 : Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• 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 33/566 : using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Abstract

The invention provides protein scaffolds and methods of preparing, screening, engineering and using such protein scaffolds.

Claims

1. A recombinant polypeptide scaffold comprising:

I. seven beta strand domains designated A, B, C, D, E, F, and G, having the amino acids of;

a) SEQ ID NO;

239 for the A beta strand, SEQ ID NO;

229 for the B beta strand, SEQ ID NO;

230 for the C beta strand, SEQ ID NO;

236 for the D beta strand, SEQ ID NO;

232 for the E beta strand, SEQ ID NO;

245 for the F beta strand and SEQ ID NO;

246 for the G beta strand;

or

b) SEQ ID NO;

239 for the A beta strand, SEQ ID NO;

229 for the B beta strand, SEQ ID NO;

247 for the C beta strand, SEQ ID NO;

236 for the D beta strand, SEQ ID NO;

232 for the E beta strand, SEQ ID NO;

248 for the F beta strand, and SEQ ID NO;

234 for the G beta strand;

II. linked to six loop regions such that a loop region connects each beta strand domain, is designated AB, BC, CD, DE, EF, and FG and corresponds to the cognate loop regions of SEQ ID NO;

1, the cognate loop regions of SEQ ID NO;

1 consisting of residues;

a) from 11 to 17 inclusive in an AB loop;

b) from 23 to 31 inclusive in an BC loop;

c) from 39 to 45 inclusive in an CD loop;

d) from 51 to 56 inclusive in an DE loop;

e) from 23 to 31 inclusive in an EF loop;

f) from 23 to 31 inclusive in an FG loop,

at least one of said loop regions being a non-naturally occurring variant of the cognate loop region in SEQ ID NO;

1; and

III. a disulfide bond linking the F and G beta strand domains or linking the C and F beta strand domains,

wherein said scaffold binds a target.

2. The scaffold of claim 1, wherein said BC loop is a non-naturally occurring variant and consists of 9 amino acids having a sequence of S-X-a-X-b-X-X-X-G, wherein X represents any amino acid, wherein (a) represents proline or alanine and wherein (b) represents alanine or glycine.

3. The scaffold of claim 1, wherein said BC loop is a non-naturally occurring variant and consists of 11 amino acids having a sequence of S-P-c-X-X-X-X-X-X-T-G (SEQ ID NO:

258), wherein X represents any amino acid and wherein (c) represents proline, serine or glycine.

4. The scaffold of claim 1, wherein said BC loop is a non-naturally occurring variant and consists of 12 amino acids having a sequence of A-d-P-X-X-X-e-f-X-I-X-G (SEQ ID NO:

257), wherein X represents any amino acid, wherein (d) represents proline, glutamate or lysine, wherein (e) represents asparagine or glycine, and wherein (f) represents serine or glycine.

5. The scaffold of claim 1, wherein said FG loop is a non-naturally occurring variant and consists of 9 amino acids having a sequence of X-a-X-X-G-X-X-X-S, wherein X represents any amino acid and wherein (a) represents asparagine, threonine or lysine.

6. The scaffold of claim 1, wherein said FG loop is a non-naturally occurring variant and consists of 10 amino acids having a sequence of X-a-X-X-X-X-b-N-P-A, wherein X represents any amino acid, wherein (a) represents asparagine, threonine or lysine and wherein (b) represents serine or glycine.

7. The scaffold of claim 1, wherein said FG loop is a non-naturally occurring variant and consists of 11 amino acids having a sequence of X-a-X-X-G-X-X-S-N-P-A (SEQ ID NO:

259), wherein X represents any amino acid, and wherein (a) represents asparagine, threonine or lysine.

8. The scaffold of claim 1, wherein said DE loop is a non-naturally occurring variant and consists of 6 amino acids having a sequence of X-X-X-X-X-X, wherein X represents any amino acid.

9. The scaffold of claim 1, wherein said AB loop is a non-naturally occurring variant and consists of 7 residues having the sequence:

K-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein (a) represents serine, threonine, alanine, or glycine.

10. The scaffold of claim 1, wherein said AB loop is a non-naturally occurring variant and consists of 9 residues having the sequence:

K-X-X-X-X-X-X-X-a, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, and wherein (a) represents serine, threonine, alanine, or glycine.

11. The scaffold of claim 1, wherein said CD loop is a non-naturally occurring variant and consists of 7, 8, or 9 residues wherein each residue in the CD loop is randomized and wherein each residue may be asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine.

12. The scaffold of claim 1, wherein said EF loop is a non-naturally occurring variant and consists of 8 residues having the sequence X-b-L-X-P-X-c-X, wherein X represents asparagine, aspartic acid, histidine, tyrosine, isoleucine, valine, leucine, phenylalanine, threonine, alanine, proline, or serine, wherein (b) represents asparagine, lysine, arginine, aspartic acid, glutamic acid, or glycine, and wherein (c) represents isoleucine, threonine, serine, valine, alanine, or glycine.

13. The scaffold of claim 1, wherein said beta strand domains have the amino acid sequences of:

SEQ ID NO;

239 for the A beta strand, SEQ ID NO;

229 for the B beta strand, SEQ ID NO;

230 for the C beta strand, SEQ ID NO;

236 for the D beta strand, SEQ ID NO;

232 for the E beta strand, SEQ ID NO;

245 for the F beta strand and SEQ ID NO;

246 for the G beta strand.

14. The scaffold of claim 1, wherein said beta strand domains have the amino acids of:

SEQ ID NO;

239 for the A beta strand, SEQ ID NO;

229 for the B beta strand, SEQ ID NO;

247 for the C beta strand, SEQ ID NO;

236 for the D beta strand, SEQ ID NO;

232 for the E beta strand, SEQ ID NO;

248 for the F beta strand and SEQ ID NO;

234 for the G beta strand.

15. The scaffold of claim 1, wherein said BC and FG loops are non-naturally occurring variants.

16. The scaffold of claim 1, wherein said BC, DE and FG loops are non-naturally occurring variants.

17. The scaffold of claim 1, wherein the target is TRAIL-R2.