AFFINITY-BASED DETECTION OF LIGAND-ENCODED SYNTHETIC BIOMARKERS

• US 20140363833A1
• Filed: 06/06/2014
• Published: 12/11/2014
• Est. Priority Date: 06/07/2013
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Bhatia, Sangeeta N., Kwong, Gabriel A., Wood, David K., Warren, Andrew D., Lin, Kevin Y.
• Granted Patent: US 10,527,619 B2
• Time in Patent Office: Days
• US Class Current: 435/7.94
• CPC Class Codes: G01N 2333/974 Thrombin
• CPC Class Codes: G01N 2800/226 Thrombotic disorders, i.e. thrombo-embolism irrespective of location/organ involved, e.g. renal vein thrombosis, venous thrombosis
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

The invention relates to methods and products associated with in vivo enzyme profiling. In particular, biomarker nanoparticles capable of quantitatively detecting enzymatic activity in vivo are described. These nanoparticles can be used to detect in vivo enzyme activity. The invention also relates to products, kits, and databases for use in the methods of the invention.

Claims

1. A method comprising

administering to a subject a biomarker nanoparticle, wherein the biomarker nanoparticle comprises a modular structure having a carrier domain linked to an enzyme susceptible detectable marker, wherein the enzyme susceptible detectable marker is comprised of an enzyme susceptible domain linked to a detectable marker whereby the detectable marker is capable of being released from the biomarker nanoparticle when exposed to an enzyme and wherein the detectable marker comprises a capture ligand and a detection ligand connected by a linker;

identifying a biological sample for detection of the marker, wherein the biological sample is at a body site that is remote from a body site where the detectable marker is released from the biomarker nanoparticle; and

,

analyzing the biological sample using a capture assay in order to detect the presence of the detectable marker, wherein the presence of the detectable marker in the biological sample is indicative of the enzyme being present in an active form within the subject.

2. The method of claim 1, wherein the capture ligand is linked to the enzyme susceptible domain.

3. The method of claim 1, wherein the detection ligand is linked to the enzyme susceptible domain.

4. The method of claim 1, wherein the capture assay involves binding of the capture ligand to an affinity agent.

10. The method of claim 1, wherein the detection ligand is a protein, a peptide, a polysaccharide, a nucleic acid, a fluorescent molecule, or a small molecule.

13. The method of claim 4, wherein the affinity agent is selected from the group consisting of antibodies, antibody fragments, aptamers, magnetic beads conjugated with Abs, proteins or peptides on an affinity column.

17. The method of claim 1, wherein the biomarker nanoparticle is administered intravenously, orally, transdermally or through an implant.

18. The method of claim 1, the carrier is a protein, a peptide or a polysaccharide.

19. The method of claim 1, the carrier is a synthetic polymer.

22. The method of claim 1, further comprising collecting the biological sample from the subject, wherein the biological sample is urine, blood, saliva, or mucous secretion.

23. The method of claim 1, wherein a plurality of biomarker nanoparticles having a plurality of enzyme susceptible detectable markers are administered to the subject.

24. The method of claim 1, wherein the biomarker nanoparticle has a plurality of enzyme susceptible detectable markers.

25. The method of claim 24, wherein the plurality of enzyme susceptible detectable markers comprise a plurality of capture ligands and a single type of detection ligand.

26. The method of claim 24, wherein the plurality of capture ligands include 2-1,000 different capture ligands.

27. The method of claim 24, wherein the plurality of capture ligands include 2-100 different capture ligands.

35. The method of claim 1, wherein the analysis method is a multiplex analysis method.

36. The method of claim 1, further comprising a purification step, wherein the detectable marker is isolated from other components in the biological sample.

39. The method of claim 1, further comprising administering to the subject a free reporter in order to normalize the detection signal.

5-9. -9. (canceled)

11-12. -12. (canceled)

14-16. -16. (canceled)

20-21. -21. (canceled)

28-34. -34. (canceled)

37-38. -38. (canceled)

40. A method comprising

collecting a urine sample from a subject suspected of having a disorder or condition, wherein the subject has been administered a biomarker nanoparticle, wherein the biomarker nanoparticle comprises a modular structure having a carrier domain linked to an enzyme susceptible detectable marker, wherein the enzyme susceptible detectable marker is comprised of an enzyme susceptible domain linked to a detectable marker whereby the detectable marker is capable of being released from the biomarker nanoparticle when exposed to an enzyme and wherein the detectable marker comprises a capture ligand and a detection ligand connected by a linker;

and,

subjecting the urine sample to analysis method by a capture assay, wherein the capture assay involves an affinity step where an affinity agent binds to the capture ligand and a detection step, wherein the detection ligand is detected in order to detect the presence of the ligand marker, and wherein the presence or absence of the ligand marker in the biological sample is indicative of the disorder or condition within the subject.

41-43. -43. (canceled)

44. A composition comprising

a transdermal patch having a drug delivery compartment, wherein the drug delivery compartment includes a biomarker nanoparticle, wherein the biomarker nanoparticle comprises a modular structure having a carrier domain linked to an enzyme susceptible detectable marker that is capable of being released from the biomarker nanoparticle when exposed to an enzyme.

45-58. -58. (canceled)

59. A method comprising

administering to a subject a microdose of a biomarker nanoparticle, wherein a detectable marker is released from the biomarker nanoparticle when exposed to an enzyme;

and analyzing a biological sample using a capture assay in order to detect the presence of the detectable marker, wherein the presence of the detectable marker in the biological sample is indicative of the enzyme being present in an active form within the subject.

60-64. -64. (canceled)

65. An article comprising

a housing comprising a membrane having a defined region with a detection reagent bound thereto, a reservoir capable of housing a biological sample in contact with the membrane such that the biological sample can be delivered to and move along the membrane, a conjugate pad on the membrane, wherein an affinity agent for binding to a capture ligand is associated with the conjugate pad, wherein the biological sample does not include a pregnancy protein.

66-71. -71. (canceled)