Multivalent constructs for therapeutic and diagnostic applications

1. An ultrasound contrast agent comprising a gas-filled microvesicle conjugated, optionally via a linker, to at least one multivalent compound comprising at least two polypeptides having specificity for different epitopes on a protein-tyrosine kinase receptor wherein said multivalent compound comprises a dimer selected form the group consisting of:

2. An ultrasound contrast agent comprising material used to form a gas-filled microvesicle conjugated, optionally via a linker, to at least one multivalent compound comprising at least two polypeptides having specificity for different epitopes on a protein-tyrosine kinase receptor wherein said multivalent compound comprises a dimer selected form the group consisting of:

3. The agent of claim 2 , wherein the material used to form a the gas-filled microvesicle comprises one or more phospholipids.

4. The agent of either of claim 1 or 2 , wherein the affinity of the multivalent compound for the protein-tyrosine kinase receptor is about 60 fold greater than the affinity of any one of the polypeptides for the target.

5. The agent of either of claim 1 or 2 , wherein the affinity of the multivalent compound for protein-tyrosine kinase receptor is about 560 fold greater than the affinity of any one of the polypeptides for the target.

6. The agent of any one of claim 1 , 2 or 3 , wherein the polypeptides have specificity for different epitopes on KDR.

7. The agent of either of any one of claim 1 , 2 or 3 , wherein the polypeptides have specificity for different epitopes on cMet.

8. The agent of claim 7 , wherein the polypeptides are independently selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, SEQ NO: 28, and SEQ ID NO: 29.

9. The agent of claim 6 , wherein at least one polypeptide comprises an amino acid substitution, and amide bond substitution, a D-amino acid substitution, a glycosylated amino acid, a disulfide mimetic substitution, an amino acid translocation, a retroinverso peptide, a peptoid, a retro-inverso peptoid, or a synthetic peptide and maintains its ability to bind the receptor.

10. The agent of claim 8 , wherein at least one polypeptide comprises an amino acid substitution, and amide bond substitution, a D-amino acid substitution, a glycosylated amino acid, a disulfide mimetic substitution, an amino acid translocation, a retroinverso peptide, a peptoid, a retro-inverso peptoid, or a synthetic peptide and maintains its ability to bind the receptor.

11. The agent of claim 6 , wherein the polypeptides comprise the sequence SEQ ID NO:11 and SEQ ID NO:5.

12. The agent of either of claim 1 or 2 , further comprising a linker between each multivalent compound and the microvesicle.

13. The agent of claim 12 , wherein the linker between each multivalent compound and the microvesicle is selected from the group consisting of a substituted alkyl chain, an unsubstituted alkyl chain, a polyethylene glycol derivative, an amino acid spacer, a sugar, an aliphatic spacer, an aromatic spacer, a lipid molecule, or combination thereof.

14. The agent of either of claim 1 or 2 , wherein the gas-filled microvesicle is selected from the group consisting of a microbubble, a microballoon, and a microsphere.

15. The agent of claim 14 , wherein the microbubble comprises a phospholipid.

16. The agent of claim 1 , wherein the microvesicle comprises a biocompatible gas, or a mixture of biocompatible gases or a gas precursor.

17. The agent of claim 16 , wherein the gas or gas mixture comprises a fluorinated gas.

18. The agent of claim 16 , wherein the gas or gas mixture comprises at least one gas selected from the group consisting of air; nitrogen; oxygen; carbon dioxide; argon, xenon; krypton; a low molecular weight alkane, cycloalkane, alkene or alkyne; a hyperpolarized gas, SF 6 , a freon and a perfluorocarbon.

19. The agent of claim 16 , wherein the gas or gas mixture comprises a gas selected from the group consisting of SF 6 , C 3 F 8 , C 4 F 8 , C 4 F 10 , and C 5 F 12 .

20. The agent of claim 15 , wherein the gas or gas mixture comprises a gas selected from the group consisting of SF 6 , C 3 F 8 , C 4 F 8 , C 4 F 10 , and C 5 F 12 .

21. The ultrasound contrast agent of claim 2 , further comprising a biocompatible gas, a mixture of biocompatible gases or a gas precursor.

22. The agent of claim 21 , wherein the gas or gas mixture comprises a fluorinated gas.

23. The ultrasound agent of claim 21 , wherein the gas or gas mixture comprises at least one gas selected from the group consisting of air; nitrogen; oxygen; carbon dioxide; argon, xenon; krypton; a low molecular weight alkane, cycloalkane, alkene or alkyne; a hyperpolarized gas, SF 6 , a freon and a perfluorocarbon.

24. The agent of claim 21 , wherein the gas or gas mixture comprises a gas selected from the group consisting of SF 6 , C 3 F 8 , C 4 F 8 , C 4 F 10 , and C 5 F 12 .

25. The agent of any one of claim 1 , 2 or 3 , further comprising a therapeutic agent.

26. The agent of claim 1 , wherein the gas-filled microvesicle comprises a microvesicle-forming material conjugated to at least one multivalent compound comprising at least two polypeptides having specificity for different epitopes on a protein-tyrosine kinase receptor, optionally via a linker wherein said multivalent compound comprises a dimer selected from the group consisting of D1, D4, D5, D6, D7, D9, D10, D11, D12, D13, D14, D15, D16, D17, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30 and D31.

27. The agent of claim 26 , wherein the microvesicle-forming material is selected from the group consisting of surfactants, lipids, sphingolipids, oligolipids, phospholipids, proteins, polypeptides, carbohydrates and synthetic or natural polymeric materials.

28. The agent of claim 2 , wherein the microvesicle-forming material is selected from the group consisting of surfactants, lipids, sphingolipids, oligolipids, phospholipids, proteins, polypeptides, carbohydrates and synthetic or natural polymeric materials.

29. A method of ultrasound imaging comprising the steps of:

(a) administering an ultrasound contrast agent of any one of claim 1 , 2 or 3 to a patient; and

(b) imaging the agent using ultrasound energy.

30. A method of imaging protein-tyrosine kinase receptors comprising the steps of:

(a) administering an ultrasound contrast agent of any one of claim 1 , 2 or 3 to a patient;

(b) allowing the contrast agent to localize at protein-tyrosine kinase receptors; and

(c) imaging the receptors using ultrasound energy.

31. The method of claim 30 , wherein the protein-tyrosine kinase receptor is selected from the group consisting of KDR and cMet.

32. A method of imaging a site of angiogenesis comprising the steps of:

(a) administering an ultrasound contrast agent of claim 6 to a patient;

(b) allowing the contrast agent to localize at sites of angiogenesis; and

(c) imaging a site of angiogenesis using ultrasound energy.

33. A method of imaging a site of hyperproliferation comprising the steps of:

(a) administering an ultrasound contrast agent of claim 7 to a patient;

allowing the contrast agent to localize at sites of hyperproliferation; and

(b) imaging a site of hyperproliferation using ultrasound energy.