Engineered integrin binding peptide compositions

Abstract

Engineered peptides that bind with high affinity (low equilibrium dissociation constant (Kd)) to the cell surface receptors of fibronectin (α 5 β 1 ) or vitronectin (α v β 3 and α v β 5 integrins) are disclosed as useful as imaging tissue. 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.

1 .- 20 . (canceled) 
     
     
         21 . A method comprising administering a therapeutically effective amount of an integrin binding peptide to an individual in need thereof,
 wherein the integrin binding peptide comprises a knottin protein scaffold comprising an engineered integrin binding loop that binds to at least one of αvβ5 integrin, αvβ3 integrin and α5β1 integrin, and   wherein the integrin binding peptide comprises an amino acid sequence at least 90% identical to the amino acid sequence of a peptide of any one of SEQ ID NO:23 through SEQ ID NO:52.   
     
     
         22 . The method according to  claim 21 , wherein the integrin binding peptide is administered parenterally to the individual. 
     
     
         23 . The method according to  claim 21 , wherein the integrin binding peptide is administered orally to the individual. 
     
     
         24 . The method according to  claim 21 , wherein the individual has a proliferative disease. 
     
     
         25 . The method according to  claim 24 , wherein the proliferative disease is cancer. 
     
     
         26 . The method according to  claim 21 , wherein the integrin binding peptide is administered to block angiogenesis in the individual. 
     
     
         27 . The method according to  claim 21 , wherein the integrin binding peptide is administered to promote cell adhesion in the individual. 
     
     
         28 . The method according to  claim 21 , wherein the integrin binding peptide comprises an amino acid sequence at least 95% identical to the amino acid sequence of a peptide of any one of SEQ ID NO:23 through SEQ ID NO:52. 
     
     
         29 . The method according to  claim 21 , wherein the integrin binding peptide comprises the amino acid sequence of a peptide of any one of SEQ ID NO:23 through SEQ ID NO:52. 
     
     
         30 . The method according to  claim 21 , wherein the integrin binding peptide comprises the amino acid sequence of the peptide of SEQ ID NO:49. 
     
     
         31 . The method according to  claim 21 , wherein the integrin binding peptide comprises the amino acid sequence of the peptide of SEQ ID NO:50. 
     
     
         32 . The method according to  claim 21 , wherein the integrin binding peptide is conjugated to an agent. 
     
     
         33 . The method according to  claim 32 , wherein the agent is a chemotherapeutic agent. 
     
     
         34 . The method according to  claim 32 , wherein the agent is a half-life extending moiety. 
     
     
         35 . The method according to  claim 34 , wherein the half-life extending moiety is polyethylene glycol (PEG). 
     
     
         36 . The method according to  claim 32 , wherein the agent is an imaging agent. 
     
     
         37 . The method according to  claim 36 , wherein the imaging agent is a positron emission tomography (PET)-based imaging agent. 
     
     
         38 . The method according to  claim 37 , wherein the (PET)-based imaging agent comprises  18 F or  64 Cu. 
     
     
         39 . The method according to  claim 36 , wherein the imaging agent is a single photon emission computed tomography (SPECT) imaging agent. 
     
     
         40 . The method according to  claim 39 , wherein the SPECT imaging agent is Indium-111, technetium-99m, or lodine-131. 
     
     
         41 . A method of imaging a tissue highly expressing an endothelial integrin that is at least one of αvβ5 integrin, αvβ3 integrin and α5β1 integrin, comprising contacting said tissue with an integrin binding peptide,
 wherein said integrin binding peptide comprises a knottin protein scaffold comprising an engineered integrin binding loop that binds to at least one of αvβ5 integrin, αvβ3 integrin and α5β1 integrin, 
 wherein said integrin binding peptide comprises an amino acid sequence at least 90% identical to the amino acid sequence of a peptide of any one of SEQ ID NO:23 through SEQ ID NO:52, and 
 wherein said integrin binding peptide further comprises a label that can be detected when the integrin binding peptide is bound to the tissue, thereby imaging the tissue. 
 
     
     
         42 . An integrin binding peptide or a pharmaceutical composition comprising said integrin binding peptide,
 wherein said integrin binding peptide comprises a knottin protein scaffold comprising an engineered integrin binding loop that binds to at least one of αvβ5 integrin, αvβ3 integrin and α5β1 integrin, and   wherein said integrin binding peptide comprises an amino acid sequence at least 90% identical to the amino acid sequence of a peptide of any one of SEQ ID NO:23 through SEQ ID NO:52.