Modified polypeptides stabilized in a desired conformation and methods for producing same

• US 20050260192A1
• Filed: 03/15/2005
• Published: 11/24/2005
• Est. Priority Date: 09/01/2000
• Status: Active Grant

• Current Assignee: Center For Blood Research Incorporated
• Original Assignee: Center For Blood Research Incorporated
• Inventors: Lu, Chafen, Shimaoka, Motomu, Springer, Timothy A.
• Granted Patent: US 7,241,869 B2
• Time in Patent Office: Days
• US Class Current: 424/130.100
• 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 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 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• 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 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• 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 37/02 Immunomodulators
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• 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 14/70546 Integrin superfamily
• CPC Class Codes: Y10S 930/26 Containing cys-cys disulfide bridge between nonadjacent cysteine residues

Abstract

The present invention provides a method for stabilizing a protein in a desired conformation by introducing at least one disulfide bond into the polypeptide. Computational design is used to identify positions where cysteine residues can be introduced to form a disulfide bond in only one protein conformation, and therefore lock the protein in a given conformation. Accordingly, antibody and small molecule therapeutics are selected that are specific for the desired protein conformation. The invention also provides modified integrin I-domain polypeptides that are stabilized in a desired conformation. The invention further provides screening assays and therapeutic methods utilizing the modified integrin I-domains of the invention.

Claims

1. :

A modified integrin I-domain polypeptide containing at least one disulfide bond, such that said modified I-domain polypeptide is stabilized in a desired conformation.

2. :

A modified integrin I-domain polypeptide of claim 1 which is stabilized in the open conformation.

3. :

A modified integrin I-domain polypeptide of claim 1 which is stabilized in the closed conformation.

4. :

A modified integrin I-domain polypeptide of claim 2 which binds ligand with high affinity.

5. :

A modified integrin I-domain polypeptide of claim 1 which is encoded by an amino acid sequence containing at least one cysteine substitution as compared to the wild-type sequence.

6. :

A modified integrin I-domain polypeptide of claim 2, wherein the distance between C carbons of the residues that are substituted fro cysteines is 3.00-8.09 Å

.

8. :

A modified integrin I-domain polypeptide of claim 2 which is derived from the I-domain of the α

L subunit of LFA-1.

9. :

A modified integrin I-domain polypeptide of claim 3 which is derived from the I-domain of the α

L subunit of LFA-1.

10. :

A modified integrin I-domain polypeptide of claim 1 which contains amino acid substitutions selected from the group consisting of K287C/K294C, E284C/E301C, L161C/F299C, K160C/F299C, and L161C/T300C.

11. :

A modified integrin I-domain polypeptide of claim 8 which contains amino acid substitutions L289C/K294C

16. :

A modified integrin I-domain polypeptide of claim 1 which is comprised within an integrin α

subunit.

17. :

A modified integrin I-domain polypeptide of claim 16 which is further associated with an integrin β

subunit.

18. :

A modified integrin I-domain polypeptide of claim 1 which is a soluble polypeptide.

19. :

A modified integrin I-domain polypeptide of claim 1 which is operatively linked to a heterologous polypeptide.

21. :

A composition comprising a modified integrin I-domain polypeptide of claim 1 and a pharmaceutically acceptable carrier.

23. :

A composition of claim 21, further comprising an anti-inflammatory or immunosuppressive agent.

7. (canceled)

12-15. -15. (canceled)

20. (canceled)

22. (canceled)

24-72. -72. (canceled)