Multimeric tie 2 agonists and uses thereof in stimulating angiogenesis

Abstract

The present invention provides a multimeric form of a Tie 2 binding peptide monomer, wherein the multimeric form has Tie 2 agonist activity. The multimeric form, preferably a tetramer, stimulates angiogenesis and promotes wound healing. The present invention also features pharmaceutical compositions comprising the multimeric Tie 2 agonists, including those suitable for topical or systemic administration. Methods of using the multimeric Tie 2 agonists of the invention for stimulating angiogenesis and for promoting healing of wounds, such as diabetic ulcers or skin grafts, are also provided.

1 - 79 . (canceled) 
     
     
         80 . A composition comprising a multimeric form of a Tie 2 binding peptide monomer, wherein the multimeric form has Tie 2 agonist activity. 
     
     
         81 . The composition of  claim 80 , wherein the Tie 2 binding peptide monomer binds to Tie 2 with high affinity but does not substantially inhibit binding of an angiopoietin to Tie 2. 
     
     
         82 . The composition of  claim 81 , wherein the angiopoietin is Ang 1. 
     
     
         83 . The composition of  claim 80 , wherein the Tie 2 binding peptide monomer comprises an amino acid sequence that is not present in a native Tie 2 ligand. 
     
     
         84 . The composition of  claim 80 , wherein the Tie 2 binding peptide monomer comprises a T7 peptide (SEQ ID NOs: 1 or 2). 
     
     
         85 . The composition of  claim 80 , wherein the Tie 2 binding peptide monomer comprises a GA3 peptide (SEQ ID NOs: 3 or 4). 
     
     
         86 . The composition of  claim 80 , wherein the Tie 2 binding peptide monomer comprises a peptide selected from the group consisting of a T4 peptide (SEQ ID NOs: 9-10), a T6 peptide (SEQ ID NOs: 7-8) and a T8 peptide (SEQ ID NOs: 5-6). 
     
     
         87 . The composition of  claim 80 , wherein the multimeric form is a tetramer. 
     
     
         88 . The composition of  claim 80 , wherein the multimeric form is a dimer. 
     
     
         89 . The composition of  claim 80 , wherein the multimeric form comprises six, eight, ten or twelve units of the Tie 2 binding peptide monomer. 
     
     
         90 . The composition of  claim 80 , wherein the Tie 2 binding peptide monomer comprises a structure: A-B-C, wherein A comprises a Tie 2 binding peptide, B comprises a spacer and C comprises a multimerizing group, wherein C has affinity for D, a multimer agent comprising multiple binding sites for C. 
     
     
         91 . The composition of  claim 90 , wherein the multimer agent D has four binding sites for the multimerizing group C such that a tetramer is formed when four Tie 2 binding peptide monomers, A-B-C, interact with the multimer agent D. 
     
     
         92 . The composition of  claim 80 , wherein the Tie 2 binding peptide monomer comprises a structure: A-B, wherein A comprises a Tie 2 binding peptide and B comprises a spacer, wherein the multimeric form is created by covalent linkage of multiple Tie 2 binding peptide monomers via the spacer B. 
     
     
         93 . The composition of  claim 80 , which comprises a tetramer form of a Tie 2 binding peptide monomer, wherein the Tie 2 binding peptide monomer comprises a structure: A-B-C, wherein:
 A comprises a Tie 2 binding peptide selected from a T7 peptide (SEQ ID NOs: 1 or 2) and a GA3 peptide (SEQ ID NOs: 3 or 4);   B comprises a polyethylene glycol spacer; and   C comprises a biotin group,   
       wherein four copies of ABC are associated with a tetramer agent, D, to create the tetramer form, the tetramer agent, D, being selected from the group consisting of avidin, streptavidin and neutravidin. 
     
     
         94 . The composition of  claim 80 , which comprises a peptide dimer, comprising: (a) a first peptide chain; (b) a second peptide chain; and (c) a linking moiety connecting said first and second peptide chains, wherein said peptide dimer binds to and activates the Tie 2 receptor. 
     
     
         95 . The composition of  claim 94 , wherein the first peptide chain and the second peptide chain are both T7 peptides (SEQ ID NOs: 1 or 2). 
     
     
         96 . The composition of  claim 94 , wherein the linking moiety comprises one or more water soluble polymers covalently bound to the first peptide chain and the second peptide chain. 
     
     
         97 . The composition of  claim 96 , wherein the one or more water soluble polymers are linear polymers. 
     
     
         98 . The composition of  claim 97 , wherein the water soluble polymer is a polyethylene glycol (PEG). 
     
     
         99 . The composition of  claim 80 , which comprises a peptide tetramer, comprising: (a) a first peptide chain; (b) a second peptide chain; (c) a third peptide chain; (d) a fourth peptide chain; and (e) a linking moiety connecting said first, second, third and fourth peptide chains, wherein said peptide tetramer binds to and activates the Tie 2 receptor. 
     
     
         100 . The composition of  claim 99 , wherein the first, second, third and fourth peptide chains are T7 peptides (SEQ ID NOs: 1 or 2). 
     
     
         101 . The composition of  claim 99 , wherein the linking moiety comprises one or more soluble polymers covalently bound to the first, second, third and fourth peptide chains. 
     
     
         102 . The composition of  claim 101 , wherein the water soluble polymer is a branched chain water soluble polymer. 
     
     
         103 . The composition of  claim 102 , wherein the water soluble polymer is a branched chain polyethylene glycol (PEG). 
     
     
         104 . A composition comprising a Tie 2 binding peptide monomer, the Tie 2 binding peptide monomer comprising a structure AB-C, wherein:
 A comprises a Tie 2 binding peptide;   B comprises a spacer; and   C comprises a multimerizing group.   
     
     
         105 . A method of stimulating angiogenesis at a site in a subject comprising contacting the site with the multimeric form of a Tie 2 binding peptide monomer of  claim 80  such that angiogenesis is stimulated at the site in the subject. 
     
     
         106 . The method of  claim 105 , wherein angiogenesis stimulated by the multimeric form is characterized by at least one of the following properties:
 a) recruitment of perivascular support cells;   b) non-leakiness of vessels; and   c) well-defined arborization.   
     
     
         107 . The method of  claim 105 , which further comprises contacting the site in the subject with a second angiogenic agent. 
     
     
         108 . The method of  claim 105 , wherein angiogenesis is stimulated in a clinical situation selected from the group consisting of vascularization of regenerative tissues, ischemic limb disease, cerebral ischemia, conditions of vascular inflammation, arteriosclerosis, avascular necrosis, stimulation of hair growth and erectile dysfunction. 
     
     
         109 . A method of decreasing vascular permeability at a site of leaky vessels comprising contacting the site of leaky vessels with the multimeric form of the Tie 2 binding peptide monomer of  claim 80  such that vascular permeability is decreased.