Affinity-based detection of ligand-encoded synthetic biomarkers

• US 10,527,619 B2
• Filed: 06/06/2014
• Issued: 01/07/2020
• 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., Wood, David K., Kwong, Gabriel A., Warren, Andrew D., Lin, Kevin Y.
• Primary Examiner(s): Cook, Lisa V
• Application Number:
  US14/298,662
• Publication Number:
  US 20140363833A1
• Time in Patent Office: 2,041 Days
• Field of Search: None
• 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 synthetic particle comprising a carrier domain linked by an enzyme susceptible substrate to a detectable marker that comprises a capture ligand that is a hapten connected through a GluFib peptide to a detection ligand,

wherein, when the synthetic particle encounters an enzyme within the subject resulting in in vivo cleavage of the enzyme susceptible substrate, the detectable marker is released from the carrier domain to renal clearance into urine;

obtaining a sample from the subject; and

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

2. The method of claim 1, wherein the synthetic particle has a plurality of detectable markers.

3. The method of claim 1, wherein the capture assay comprises an ELISA assay or a lateral-flow assay (LFA).

4. The method of claim 3, wherein the enzyme comprises thrombin or matrix-metalloprotease 9 (MMP9).

5. The method of claim 4, wherein the detectable marker comprises the capture ligand at a C-terminus of the GluFib peptide and the detection ligand at an N-terminus of the GluFib peptide.

6. The method of claim 5, wherein when the synthetic particle does not encounter an enzyme resulting in in vivo cleavage of the enzyme susceptible substrate, the synthetic particle is cleared from the subject via organs of the reticuloendothelial system (RES).

7. The method of claim 5, the carrier domain is a protein, a peptide, a polysaccharide, or a synthetic polymer.

8. The method of claim 7, wherein the synthetic particle has a size such that it may only be cleared from the subject by organs of the RES, and the detectable marker has a size such that it is cleared from the subject into urine by renal clearance.

9. The method of claim 1, wherein the synthetic particle has a size such that it may only be cleared from the subject by organs of the RES, and the detectable marker has a size such that it is cleared from the subject into urine by renal clearance.

10. The method of claim 1, wherein the capture assay comprises an affinity agent linked to a solid support wherein the affinity agent binds to the capture ligand on the detectable marker in the urine sample.

11. The method of claim 10, wherein the affinity agent comprises an antibody.

12. The method of claim 10, 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.

13. The method of claim 1, wherein the synthetic particle is administered intravenously, orally, or transdermally.

14. The method of claim 1, wherein a plurality of synthetic particles having a plurality of detectable markers are administered to the subject.

15. The method of claim 1, wherein the synthetic particle has a plurality of detectable markers.

16. The method of claim 15, wherein the plurality of enzyme susceptible detectable markers comprise a plurality of capture ligands and a single type of detection ligand.

20. The method of claim 1, wherein the hapten is selected from the group consisting of fluorescein (FAM), dinitrophenyl (DNP), tetramethylrhodamine (TMR), and Alexa Fluor 488 (AF488).

21. The method of claim 1, wherein the detection ligand is biotin.

17. A method comprising:

administering to a subject a particle comprising a carrier domain linked via an enzyme cleavable linker to a detectable marker that includes a hapten, a GluFib peptide, and a detection ligand, wherein a size of the particle inhibits renal clearance and wherein the particle is cleaved in vivo by an enzyme to release the detectable marker, which admits of renal clearance and is excreted in urine;

obtaining a sample from the subject; and

assaying the sample for the detectable marker via a capture assay in which an antibody on a substrate binds to the hapten and a detectable reporter binds to the detection ligand to provide a signal indicative of the enzyme being active within the subject.

18. The method of claim 17, wherein:

the hapten includes one selected from the group consisting of fluorescein, dinitrophenyl, tetra-methylrhodamine, and Alexa Fluor 488;

the capture assay includes an ELISA assay;

the substrate includes a multi-well plate; and

the detectable reporter includes horseradish peroxidase.

19. The method of claim 17, wherein:

the hapten includes one selected from the group consisting of fluorescein, dinitrophenyl, tetra-methylrhodamine, and Alexa Fluor 488;

the capture assay includes lateral flow assay;

the substrate includes a paper strip; and

the detectable reporter includes a gold particle.

22. The method of claim 17, wherein the detection ligand is biotin.