Engineered Integrin Binding Peptides

• US 20090257952A1
• Filed: 04/03/2009
• Published: 10/15/2009
• Est. Priority Date: 10/04/2006
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: COCHRAN, Jennifer R., LAHTI, Jennifer, LEVIN, Aron M., CHENG, Zhen, GAMBHIR, Sanjiv S., KIMURA, Richard, SILVERMAN, Adam
• Granted Patent: US 8,536,301 B2
• Time in Patent Office: Days
• US Class Current: 424/1.690
• CPC Class Codes: A61K 38/06 Tripeptides
• CPC Class Codes: A61K 38/08 Peptides having 5 to 11 amino acids A61K38/043 - A61K38/046 take precedence
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino acids
• CPC Class Codes: A61K 51/082 the peptide being a RGD-containing peptide
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells

Abstract

Engineered peptides that bind with high affinity (low equilibrium dissociation constant (Kd)) to the cell surface receptors of fibronectin (α₅β₁) or vitronectin (α_vβ₃ and α_vβ₅ integrins) are disclosed. These peptides are based on a molecular scaffold into which a subsequence containing the RGD integrin-binding motif has been inserted. The subsequence (RGD mimic) comprises about 9-13 amino acids, and the RGD contained within the subsequence can be flanked by a variety of amino acids, the sequence of which was determined by sequential rounds of selection (in vitro evolution). The molecular scaffold is preferably based on a knottin, e.g., EETI (Trypsin inhibitor 2 (Trypsin inhibitor II) (EETI-II) [Ecballium elaterium (Jumping cucumber)], AgRP (Agouti-related protein), and Agatoxin IVB, which peptides have a rigidly defined three-dimensional conformation. It is demonstrated that EETI tolerates mutations in other loops and that the present peptides may be used as imaging agents.

Claims

1. An integrin binding peptide, comprising a binding sequence specific to bind to at least one of α

_vβ

₅ integrin, α

_vβ

₃ integrin and α

₅β

₁ integrin, said binding sequence being comprised in a knottin protein scaffold and being an engineered integrin binding loop between 9 and 13 amino acids long comprising the sequence RGD, and said knottin protein scaffold, except for the engineered integrin binding loop, being substantially identical to one of;

EETI-II, AgRP, mini-AGRP, agatoxin or miniagatoxin.

2. The integrin binding peptide of claim 1 having a Kd of not more than 100 nM.

3. The integrin binding peptide of claim 1 where the molecular scaffold is EETI-II.

4. The integrin binding peptide of claim 1 wherein the molecular scaffold is either AgRP, mini-AgRP, agatoxin, or mini-agatoxin.

5. The integrin binding peptide of claim 1 having a sequence at least 90% identical to a peptide listed as SEQ ID NO:

23 through SEQ ID NO;

52, inclusive.

6. The integrin binding peptide of claim 1 where the integrins bound are α

_vβ

₃ and α

vβ

5.

7. The integrin binding peptide of claim 1 where the molecular scaffold is a peptide comprising at least three cysteine disulfide linkages and having crosslinked cysteine residues adjacent the engineered integrin binding loop.

8. The integrin binding peptide of claim 7 which is substantially identical to one of peptide 1.5B (SEQ ID NO:

28), 2.5D (SEQ ID NO;

     49) or 2.5F (SEQ ID NO;

     50).

9. The integrin binding peptide of claim 1 where the molecular scaffold is a peptide substantially identical to mini-AgRP.

10. The integrin binding peptide of claim 1 where the molecular scaffold is a peptide substantially identical to agoutin or mini-agoutin.

11. The integrin binding peptide of claim 1 wherein the substantial identity is at least 90% in the molecular scaffold and at least 95% in the binding loop.

12. A vector encoding a peptide sequence according to claim 1.

13. A method of inhibiting binding of an integrin to vitronectin, comprising contacting said integrin with an integrin binding peptide according to claim 1.

14. A method of treating a proliferative disease comprising the step of administering to a subject in need thereof a composition comprising an integrin binding peptide according to claim 1.

15. A method of promoting cell adhesion comprising the step of contacting cells with an immobilized material comprising an integrin binding peptide according to claim 1.

24. A polynucleic acid, encoding an integrin binding peptide according to claim 1.

25. A eukaryotic expression vector comprising a DNA sequence of claim 24.

26. A host cell transformed with a vector according to claim 25.

16. A method of imaging tissues expressing an endothelial integrin comprising the step of contacting the tissue to be imaged with a composition comprising an integrin binding peptide comprising a binding sequence specific to bind to at least one of α

_vβ

₅ integrin, α

_vβ

₃ integrin and α

₅β

₁ integrin, said binding sequence being comprised in a knottin protein scaffold, said binding sequence being an engineered integrin binding loop between 9 and 13 amino acids long, said loop comprising the sequence RGD, and said scaffold, except for the engineered integrin binding loop, being substantially identical to one of;

EETI-II, AgRP, mini-AGRP, agatoxin or miniagatoxin.

17. The method of claim 16 wherein said integrin binding peptide is labeled with a label selected from the group consisting of:

an optical label and a positron-emitting label.

18. The method of claim 17 wherein the positron emitting label is 18F or 64Cu.

19. The method of claim 18 wherein the label is conjugated with DOTA.

20. The method of claim 19 wherein the imaging is positron emission tomography.

21. The method of claim 20 where the integrin binding has a Kd of not more than 100 nM.

22. The method of claim 21 wherein the integrin binding peptide binds to α

vβ

5 and α

vβ

3 integrins.

23. The method of claim 22 wherein the tissue is a tumor.

27. A DNA sequence encoding an engineered integrin binding peptide, substantially identical to a selected from a sequence selected from the Group consisting of SEQ ID NO:

95-104.

28. A molecular scaffold for presenting a structurally constrained loop insert, wherein the scaffold is an AgRP peptide having a sequence substantially identical to an AgRP peptide sequence as set forth in SEQ ID NO:

54.

29. An integrin binding peptide, comprising a binding sequence specific to bind to at least one of α

_vβ

₅ integrin, α

_vβ

₃ integrin and α

₅β

₁ integrin, said binding sequence being comprised in a knottin protein scaffold, said binding sequence being an engineered integrin binding loop between 9 and 13 amino acids long, said loop comprising the sequence RGD, and said scaffold, except for the engineered integrin binding loop, being substantially identical to one of;

EETI-II, AgRP, mini-AGRP, agatoxin or miniagatoxin, with the proviso that if the scaffold is AGrP, loops 1, 2, or 3 may be mutated, and if the scaffold is EETI-II, loops 2 and 3 may be mutated.